WO2024066317A1 - Method for preparing plant cell nucleus suitable for single-cell sequencing of chinese actinidia root tissue and optimizing suspension - Google Patents

Method for preparing plant cell nucleus suitable for single-cell sequencing of chinese actinidia root tissue and optimizing suspension Download PDF

Info

Publication number
WO2024066317A1
WO2024066317A1 PCT/CN2023/090396 CN2023090396W WO2024066317A1 WO 2024066317 A1 WO2024066317 A1 WO 2024066317A1 CN 2023090396 W CN2023090396 W CN 2023090396W WO 2024066317 A1 WO2024066317 A1 WO 2024066317A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
centrifuge tube
ice
kiwifruit
tissue
Prior art date
Application number
PCT/CN2023/090396
Other languages
French (fr)
Chinese (zh)
Inventor
范黎明
肖云平
Original Assignee
上海欧易生物医学科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海欧易生物医学科技有限公司 filed Critical 上海欧易生物医学科技有限公司
Publication of WO2024066317A1 publication Critical patent/WO2024066317A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention belongs to the field of biotechnology and relates to a method for preparing plant cell nuclei, and specifically relates to a method for preparing cell nuclei of kiwifruit root tissue and an application and use method thereof in plant single-cell sequencing.
  • Single-cell sequencing technology measures the transcriptome of a single cell and uses "single-cell resolution" to analyze the molecular mechanisms behind life phenomena. It is a cutting-edge technical means for life science research and has been widely used.
  • single-cell sequencing of plant tissues is "difficult". Plant cells must first be dissociated into protoplasts before single-cell sequencing can be performed.
  • the main components of the cell wall are polysaccharides such as cellulose. These macromolecules have variable structures and stable chemical properties and are one of the most difficult substances to degrade. Therefore, it is currently difficult to prepare protoplasts that meet the requirements of single-cell sequencing from most plant tissues, which greatly limits plant single-cell sequencing research.
  • the preparation of protoplasts causes very large changes to plant cells, which will induce transcriptional changes, resulting in a serious reduction in the reliability of sequencing results, and the data cannot reflect real biological changes.
  • Arabidopsis has mature cell nucleus preparation technology solutions, such as the experimental steps described in "Identification of OpenChromatin Regions in Plant Genomes Using ATAC-Seq” and “Isolation of Plant RootNuclei for Single Cell RNA Sequencing", the former is mainly used in Arabidopsis genome sequencing research, and the latter is mainly used in transcriptome sequencing research.
  • the two methods can prepare cell nucleus suspensions of Arabidopsis leaf and root tissues, respectively.
  • Kiwifruit is a large deciduous vine with very low tissue similarity to Arabidopsis, with large differences in cell wall composition, and due to different growth environments, the composition of tissues and cells is also very different.
  • the method of preparing cell nuclei using Arabidopsis cannot prepare kiwifruit root tissue cell nucleus suspensions that meet the requirements of single-cell sequencing.
  • the present invention based on a large amount of in-depth research and experiments, provides a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, as well as a corresponding lysate formula and a method for optimizing a cell nucleus suspension (wherein the cell nucleus suspension optimization method described in the present invention mainly optimizes the removal of calcium oxalate crystals deposited in kiwifruit root tissue/cells, the removal of mRNA released into the suspension during the extraction of kiwifruit root tissue, and the removal of tissue fragments in the cell nucleus suspension).
  • the method for preparing kiwifruit root tissue cell nuclei suitable for single-cell sequencing comprises the following specific steps:
  • Lyse cells After disruption, place the centrifuge tube on ice and incubate for lysis.
  • step (1) the KR lysate contains the following components (final concentration):
  • L-tartaric acid is commonly used as an acidulant for beverages and other foods. In the present invention, it serves to neutralize the cell contents released after tissue and cell disruption, stabilize the pH of the lysate, and maintain the integrity of the cell nucleus; at the same time, L-tartaric acid acts as a reducing agent in the lysate to maintain the activity of the RNase inhibitor.
  • saponin mainly functions as a pharmaceutical raw material, and is also used as a preservative material and an emulsifier component.
  • saponin as a component of a lysing solution, plays a role in lysing cell membranes and releasing cell nuclei.
  • step (1) when preparing the KR lysate, 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) needs to be added first, the solution is thoroughly mixed and placed on ice for precooling before adding 0.2 to 0.4 U/mL RNase inhibitor.
  • the purpose is that 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) can maintain the activity of 0.2 to 0.4 U/mL RNase inhibitor.
  • the precooling temperature is 0-5°C; preferably, it is precooled in a 4°C refrigerator.
  • the centrifuge tube is a 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, RNase-/DNase-free and nonpyrogenic (Axygen, MCT-200-C).
  • the crusher is commonly used in nucleic acid extraction. Its role in the present invention is to fully destroy plant tissues through steel balls, and assist KR lysis solution to complete the preparation of crude cell nucleus suspension.
  • the diameter of the steel balls is 5 mm.
  • the crusher is Wanbo Bio high-throughput tissue crusher (onebio-48p).
  • the crushing conditions are 1200-1300 rpm, 170-180 s; preferably, 1200 rpm, 180 s.
  • the kiwifruit root may refer to the root tip tissue of any variety of kiwifruit, including meristem, elongation zone, and ripening zone.
  • the fresh weight of the kiwifruit root tissue is 50-100 mg; preferably, 100 mg.
  • step (3) the purpose of the incubation is to use KR lysis solution to remove the cell membrane and other structures outside the cell nucleus at a temperature that maintains the integrity of the messenger RNA (mRNA) in the cell nucleus.
  • the incubation time is 7 to 8 minutes; preferably, 7 minutes.
  • the filter is FALCON 40 ⁇ m Cell Strainer (FALCON, 352340) with a pore size of 40 ⁇ m.
  • the PBS is Gibco PBS pH7.4 (1 ⁇ ) (Gibco, 10010-031).
  • the centrifuge tube is Corning Centristar 15mL centrifuge tube (Corning, 430790).
  • the filtration is performed at 0-5°C throughout the entire process; preferably, the operation is performed on ice. The purpose of standing on ice for 1 minute in step (4) is to allow large tissue blocks to settle to the bottom of the centrifuge tube.
  • tissue blocks will not settle completely, causing the screen to be blocked during filtration, resulting in loss of cell nuclei; if the standing time is too long, there is a risk of mRNA degradation, and the degradation of mRNA will directly lead to the failure of the experiment.
  • the tissue block sedimentation time of 1min can ensure complete sedimentation without the risk of mRNA degradation.
  • the filter is FALCON 40 ⁇ m Cell Strainer (FALCON, 352340) with a pore size of 40 ⁇ m.
  • the centrifugation temperature is 4-6°C; preferably, 4°C.
  • the centrifugation time is 10-15 minutes; preferably, 10 minutes.
  • the centrifugal force of the centrifugation is 300-500 ⁇ g, preferably, 300 ⁇ g.
  • the PBS is PBS that has been precooled in a 4°C refrigerator for 30 minutes in advance.
  • the sorting column is a Miltenyi MS Column (Miltenyi, 130-042-201).
  • the sorting column is usually used in a magnetic field to bind specific antigens on the surface of the target cell membrane through antibodies with magnetic beads, so as to be used for sorting specific cell types of animals.
  • the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized, and small cell nuclei can pass through the sorting column with the liquid flow, while the needle-shaped calcium oxalate crystals will be retained in the sorting column and cannot pass through with the liquid flow, thereby achieving the purpose of purifying cell nuclei.
  • the filtration is operated at 0-5°C throughout the whole process; preferably, it is placed on ice for operation.
  • the centrifugation temperature is 4-6°C; preferably, 4°C.
  • the centrifugation time is 10-15 minutes; preferably, 10 minutes.
  • the centrifugal force is 300-500 ⁇ g; preferably, 300 ⁇ g.
  • the PBS is PBS pre-cooled on ice.
  • the present invention also provides kiwifruit root tissue cell nuclei suitable for single-cell sequencing obtained by the above method.
  • the present invention also provides the application of the kiwifruit root tissue cell nucleus in single-cell sequencing.
  • the lysis buffer was prepared according to the following ingredients: 2%-3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.2-0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613).
  • step 6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • step (8) Microscopic examination and counting. Take 5 ⁇ L of the resuspension in step (8) and mix it with DAPI staining solution in a ratio of 1:1. Use a hemocytometer to count the total number and concentration of cell nuclei.
  • the present invention also provides a KR lysate, which comprises the following components (final concentration): 2% to 3% (v/v) L-tartaric acid, 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin, and 0.2 to 0.4 U/mL recombinant RNase inhibitor.
  • the present invention also provides a reagent/kit, characterized in that it comprises the KR lysate as described above.
  • the present invention also provides the use of the KR lysate or the reagent/kit in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, kiwifruit root tissue nuclei transcriptome sequencing, and kiwifruit root tissue nuclei ATAC sequencing research.
  • the present invention has the following beneficial effects:
  • Kiwifruit root tissue contains a large amount of impurities, polysaccharides, etc., and there are a large number of debris, intracellular crystals and other impurities in the suspension after lysis. Its quality is completely unable to meet the requirements of 10x Genomics, which seriously hinders the research progress of kiwifruit root tissue.
  • the quality of the nuclei prepared according to the technical solution of the present invention is very high (the sample quality is improved from 3 ⁇ 105 nuclei and a fragmentation rate of more than 70% to 2.26 ⁇ 106 ⁇ 4.12 ⁇ 106 and a fragmentation rate of 3 ⁇ 5%.), cell fragments, intracellular crystals and other impurities are basically completely removed, and subsequent single-cell experiments can be carried out smoothly.
  • the technical solution provided by the present invention uses only 4 lysate components, which is significantly reduced compared with 6 to 7 components in the prior art; the experimental process can complete the preparation of cell nuclei within 30 minutes. It avoids steps such as sucrose deposition, saves reagents and consumables, reduces costs, and greatly shortens time, improves efficiency, and facilitates the development of large-scale sample experiments.
  • Figure 1 is a microscopic examination of a cell nucleus.
  • the figure shows a cell nucleus prepared by the technical method of the present invention. It can be seen in the figure that impurities such as cell debris and calcium oxalate crystals are completely removed, and only a very small proportion of impurities smaller than the cell nucleus exist.
  • FIG. 2 is the cDNA quality inspection results of single-cell transcriptome sequencing of Examples 1 to 4 of the present invention (Agilent 4150 Bioanalyzer).
  • FIG. 3 is a diagram of needle-shaped calcium oxalate crystals in tissue.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (8) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • Results and analysis The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal ( Figure 2).
  • Table 1 The results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has a good effect.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (8) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • Results and analysis The results are shown in Table 1.
  • the RNA content in the suspension was extremely low.
  • the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all met the single-cell sequencing requirements of 10x Genomics.
  • the cDNA length was normal.
  • the library construction results showed that the single-cell transcriptome sequencing of the cell nucleus suspension extracted from the frozen samples was effective (Figure 2).
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (8) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • Results and analysis The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal ( Figure 2).
  • Table 1 The results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has good results.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (8) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • Results and analysis The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal ( Figure 2).
  • Table 1 The results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has good results.
  • Arabidopsis leaf nuclei were prepared according to "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq".
  • step (6) Collect the precipitate by centrifugation. Resuspend the nuclear precipitate in step (5) with 1 mL of pre-cooled nuclear extraction solution 1. Aspirate the resuspended solution and add it to a new 1.5 mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 ⁇ g and 4°C for 10 minutes. Carefully remove the centrifuge tube and aspirate and discard the supernatant.
  • step (8) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 ⁇ L of the resuspension in step (8), mix it with DAPI stain in a ratio of 1:1, use a pipette to draw 10 ⁇ L and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
  • Arabidopsis root tissue nuclei were prepared according to the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing".
  • Transferring materials Use forceps to transfer fresh root samples to a 60 mm culture dish and immerse all roots in NIB lysis solution.
  • Tissue mincing mince the root sample using a double-sided blade for about 5 minutes.
  • Tissue lysis Incubate at 4°C with gentle horizontal shaking for 15 minutes to allow lysis.
  • step (8) Microscopic examination and counting; Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope by using DAPI staining solution: Take 5 ⁇ L of the resuspension in step (8), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 ⁇ L and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
  • the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" was used to prepare the kiwifruit leaf tissue nucleus. The remaining steps were the same as those in Comparative Example 4.
  • This example uses the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" to prepare kiwifruit root tissue nuclei.
  • the remaining steps are the same as those in Comparative Example 4.
  • ST1 buffer was used to prepare kiwifruit root tissue cell nuclei.
  • Step (3) The pH of the buffer solution in step (3) was measured using a pH meter (METTLER TOLEDO FE28-CN, 30595013).
  • ST4 buffer containing 15 mM sodium citrate and 20 mM MOPS (3-(N-morpholine) propanesulfonic acid, Sigma-Aldrich, 1132-61-2) was used to prepare kiwifruit root tissue nuclei, and the remaining steps were the same as those in Comparative Example 7. The results are shown in Table 2.
  • This example uses KR1 lysis buffer to prepare kiwifruit root tissue nuclei.
  • the kiwifruit root tissue nucleus was prepared by using KR2 lysate with components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) sodium deoxycholate (Sigma, 302-95-4), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • the kiwifruit root tissue nuclei were prepared by using KR3 lysate with components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) digitonin (Sigma, 11024-24-1), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • the kiwifruit root tissue nucleus was prepared by using KR4 lysate components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (v/v) Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, Sigma, 9036-19-5), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • the kiwifruit root tissue nuclei were prepared by using KR5 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • the kiwifruit root tissue nuclei were prepared by using KR6 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.1% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • the kiwifruit root tissue nuclei were prepared by using KR7 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
  • This example uses a double-sided blade to chop kiwifruit root tissue and KR5 lysis buffer for lysis.
  • kiwifruit root tissue was cut into pieces using scissors and lysed using KR5 lysis solution, and the operation was the same as that of comparative example 17. The results are shown in Table 3.
  • kiwifruit root tissue was homogenized and broken using a glass homogenizer, and lysed using KR5 lysis buffer.
  • kiwifruit root tissue was broken using a disruptor and lysed using KR5 lysis buffer.
  • This example uses liquid nitrogen to grind and crush kiwifruit root tissue, and KR5 lysis buffer for lysis.
  • kiwifruit root tissue was broken using a disruptor and lysed using KR8 lysis buffer.
  • RNA quality inspection The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). RNA quality inspection was performed using an Agilent 2100 bioanalyzer. The results are shown in Table 4
  • kiwifruit root tissue was broken using a disruptor and lysed using KR9 lysis buffer.
  • RNA quality inspection The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). RNA quality inspection was performed using an Agilent 2100 bioanalyzer. The results are shown in Table 4
  • the kiwifruit root tissue was broken using a crusher, and the KR10 lysis solution contained 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.6 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613). The remaining operations were the same as those in Comparative Example 25. The results are shown in Table 5
  • This example uses a 70 ⁇ m (FALCON, 352350) mesh to filter the cell nucleus suspension twice.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • This example uses a 70 ⁇ m (FALCON, 352350) mesh to filter out calcium oxalate crystals.
  • This example is based on the removal of calcium oxalate crystals using 100 ⁇ g differential centrifugation.
  • KR lysis buffer Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Differential centrifugation. Add pre-cooled PBS to the filtrate in step (5) to 10 mL and centrifuge at 4°C, 100 ⁇ g, for 10 min. After centrifugation, transfer the supernatant to a new centrifuge tube and resuspend the precipitate in 3 mL of pre-cooled PBS.
  • This example is based on the use of density gradient centrifugation to remove calcium oxalate crystals.
  • KR lysis buffer Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • This example is based on the removal of calcium oxalate crystals using flow cytometry.
  • KR lysis buffer Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • This example is based on the use of an MS sorting column to remove calcium oxalate crystals.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • This example is based on the use of LS sorting columns to remove calcium oxalate crystals.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • the washing conditions in this embodiment are 100 ⁇ g, 4° C., and 10 min per wash.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step 8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 100 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • the washing conditions in this embodiment are 1000 ⁇ g, 4° C., and 10 min per wash.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 1000 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • the washing conditions in this embodiment are 300 ⁇ g, 4° C., and 10 min per wash.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • the washing conditions in this example are 300 ⁇ g, 4° C., and 10 min for three washes.
  • lysis buffer 1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
  • step (6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
  • step (10) Collect the precipitate by centrifugation. Place the centrifuge tube in step (9) in a centrifuge and centrifuge at 300 ⁇ g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 ⁇ L of ice-cold PBS.
  • the experimental sample of this example is 50 mg of Arabidopsis leaves, and the specific operation is the same as that of Example 1. The results are shown in Table 8
  • the experimental sample of this example is 50 mg of Arabidopsis roots, and the specific operation is the same as that of Example 1. The results are shown in Table 8
  • the experimental sample of this example is 50 mg corn embryo, and the specific operation is the same as that of Example 1. The results are shown in Table 8
  • the present invention describes a method suitable for sequencing single cell nuclei of kiwifruit root tissue, including a method for extracting cell nuclei from kiwifruit root tissue and a method for optimizing cell nuclei suspension.
  • the technical method provided by the present invention can introduce single cell sequencing technology into the relevant research on kiwifruit molecular mechanism and breeding, and can effectively and efficiently promote the progress of research.
  • the single cell nucleus preparation method for kiwifruit root tissue designed by the present invention mainly includes a lysate, whose components are 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613).
  • the cell nucleus is extracted from the tissue by crushing the sample and lysing the cells, and then the cell nucleus suspension with a number greater than 1 ⁇ 106, a fragmentation rate less than 10%, and a clumping rate less than 4% is obtained after the suspension is optimized by sieve filtration, centrifugal washing, etc., which meets the quality requirements of 10 ⁇ single cell sequencing for the cell nucleus suspension.
  • the purpose of using high-throughput single cell sequencing technology to study kiwifruit root tissue is achieved.
  • L-tartaric acid is commonly used as an acidulant for beverages and other foods, has a strong buffering capacity, and plays a major buffering role in the present invention; at the same time, tartaric acid also has a strong reducing property, and plays a protective role for nucleic acids in the cell nucleus in the present invention.
  • the results of the comparative example show that when the concentration of the buffer component is low, the pH of the buffer cannot be kept stable, and the nucleus is broken due to the change of the pH of the buffer; when the concentration of the buffer component is high, the nucleus agglomerates due to the high ion concentration in the buffer, resulting in experimental failure. Therefore, the buffer component for preparing the nucleus in the present invention is 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15 to 25 mM sodium citrate.
  • Extracting plant cell nuclei requires destroying the cell membrane, and the basic structure of the cell membrane is a phospholipid bilayer. It is common to use detergents to destroy the phospholipid bilayer to achieve the purpose of releasing the cell nucleus.
  • the lysate formula was explored, and experiments were conducted using lysate components with different properties and working principles.
  • Triton X-100 is a mild detergent derived from polyethylene oxide and contains an alkylphenyl hydrophobic group.
  • Saponin is composed of saponin and sugar, uronic acid or other organic acids, and saponin gives saponin the function of emulsifying lipid molecules.
  • the results of the comparative example show that when saponin is used as a lysis component, the cell nucleus yield is significantly improved compared with SDS, Triton X-100, etc.; at the same time, because saponin does not cause protein denaturation, saponin as a lysis component will not damage the cell nucleus and nuclear membrane.
  • the results of the comparative example show that when the saponin concentration is lower than 2%, the number of cell nuclei will decrease significantly, because saponin itself contains sugars and organic acids, etc. When the saponin concentration is higher than 4%, it will affect the stability of the lysis system and reduce the number of cell nuclei. Therefore, the concentration range of saponin in the lysis solution of the present invention is 2% to 4% (m/v).
  • Plant cell membranes are wrapped by cell walls, which are composed of cellulose, hemicellulose, pectin and other components. Kiwi plants are large deciduous vines, and the supporting strength of the cell walls is much higher than that of Arabidopsis. Therefore, in the method of breaking the cell walls, stronger breaking conditions are needed to help obtain more nuclei.
  • stronger breaking conditions are needed to help obtain more nuclei.
  • Mortar liquid nitrogen grinding is the most sufficient way to break tissue, but mortar grinding also damages the nucleus while breaking the cell wall.
  • the results of the comparative examples show that the crusher crushing method assists the lysate to fully release the nucleus while causing less damage to the nucleus, which is the best method for extracting the nucleus of kiwi root tissue.
  • the crude cell nucleus suspension extracted from the tissue after being treated with a lysate contains a large amount of impurities such as cell fragments and mRNA and organelles released by cell fragmentation. Under such conditions, the cell nucleus suspension has large fragments and cannot be used for single-cell sequencing experiments. The large fragments need to be removed to meet the requirements of single-cell sequencing. First, the larger tissue and cell fragments are filtered to remove them. By comparing Examples 29 to 33, the yield and impurity removal efficiency of the crude cell nucleus suspension filtered with different aperture meshes and different filtering times are verified. The results of Comparative Example 29 show that the 70 ⁇ m mesh cannot achieve the purpose of impurity removal very well.
  • the results of comparative examples 32-33 show that the 40 ⁇ m cell sieve can quickly remove impurities in the crude cell nucleus suspension.
  • the best method for purifying the crude cell nucleus suspension of kiwifruit root is to filter twice with a 40 ⁇ m cell sieve, with an impurity removal efficiency of up to 80%, and a cell nucleus yield of more than 70%. This choice not only quickly and effectively reduces the fragmentation rate, but also has a higher cell nucleus yield.
  • Calcium oxalate crystals exist in kiwifruit root tissues as needle crystals, with different thicknesses and lengths. According to microscopic observation, it can be seen that the length of calcium oxalate crystals is greater than 70 ⁇ m.
  • the effect of 70 ⁇ m cell sieve on removing calcium oxalate crystals was verified by comparative example 34. The results show that 70 ⁇ m cell sieve cannot effectively remove calcium oxalate needle crystals.
  • the effect of removing calcium oxalate crystals by 40 ⁇ m and 30 ⁇ m sieves was verified by comparative examples 35 to 36. The results show that reducing the sieve aperture still cannot remove calcium oxalate crystals.
  • the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized.
  • Small cell nuclei can pass through the sorting column with the liquid flow, while needle-shaped calcium oxalate crystals will be left in the sorting column and cannot pass through with the liquid flow.
  • the purpose of purifying cell nuclei can achieve the purpose of removing impurities while ensuring the cell nucleus yield, solving the problem of optimizing single cell nucleus suspensions.
  • the results show that all indicators of the filtrate obtained by filtering the single cell nucleus suspension through the MS sorting column meet the requirements of the 10 ⁇ Genmics platform for single-cell sequencing.
  • the free mRNA in the cell nucleus suspension needs to be removed by washing to improve the data quality.
  • the concentration of the cell nucleus suspension needs to be adjusted.
  • the washing conditions were verified. The results show that too small a centrifugal force will lead to a large loss of cell nuclei, too large a centrifugal force will lead to cell nucleus damage, and too many washing times will lead to cell nucleus fragmentation; the washing conditions are 4°C, 300 ⁇ g to 500 ⁇ g centrifugation for 10 minutes, washing twice, and the free mRNA in the cell nucleus suspension is basically removed.
  • the cell nucleus yield is higher than other methods, which is suitable for the washing conditions of kiwi root tissue cell nucleus suspension.
  • Comparative Examples 46 to 48 verified the results of preparing the nuclear suspension of Arabidopsis leaves, Arabidopsis root tips, and corn embryos using the KR lysate and the cell nucleus extraction and optimization methods in this patent. The results show that the cell nucleus preparation method in this patent, and the corresponding lysate formula and cell nucleus optimization method correspond one-to-one with kiwifruit root tissue, and the steps need to be performed in sequence to complete the preparation of the nuclear suspension of kiwifruit root tissue, which is not applicable to other species and tissues.
  • the present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing.
  • a KR lysate solution is obtained, which comprises 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATEBIOLO; the method of assisting the release of kiwifruit root nuclei is crushing with a crusher.
  • the present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, which can effectively prepare the nucleus suspension required for the single-cell transcriptome sequencing experiment of the 10x Genomics platform.
  • the method for preparing a kiwifruit nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention there is a one-to-one correspondence between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results, that is, there is a compatibility relationship between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results.
  • the kiwifruit root, KR lysate, and nucleus suspension filtration operation mentioned in Examples 1 to 4 of the present invention should be used according to the method specified in the present invention to effectively prepare the nucleus suspension, and the various indicators of the prepared nucleus suspension can meet the requirements of single-cell transcriptome sequencing.
  • the method for preparing a kiwifruit root cell nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention is scientifically rigorous in operation, highly repeatable, and can effectively and efficiently separate a cell nucleus suspension with a sufficient number, low impurity rate, and extremely low environmental RNA from kiwifruit root tissue, and the obtained cell nucleus suspension can meet various indicators of single-cell transcriptome sequencing on the 10x Genomics platform.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Provided is a method for preparing a Chinese actinidia root tissue cell nucleus suitable for single-cell sequencing. The cell nucleus prepared by means of an original lysis solution formula and a cell nucleus suspension optimization method has very high quality. Compared to a lysis solution formula with 6-7 components in the prior art, components of the provided lysis solution formula are significantly reduced. The preparation of the cell nucleus can be completed within 30 minutes of an experimental process. According to the method, the steps of sucrose deposition and the like are avoided, reagents and consumables are saved, and the cost is reduced; and meanwhile, the time is greatly shortened, and the efficiency is improved, thereby facilitating the conduct of experiments with a large number of samples. Further provided is a KR lysis solution, a reagent/kit, and use thereof in a method for preparing a Chinese actinidia root tissue cell nucleus suitable for single-cell sequencing.

Description

一种适用于猕猴桃根组织单细胞测序的植物细胞核制备以及悬液优化方法A plant nucleus preparation and suspension optimization method suitable for single-cell sequencing of kiwifruit root tissue 技术领域Technical Field
本发明属于生物技术领域，涉及植物细胞核的制备方法，具体涉及一种猕猴桃根组织的细胞核制备方法及其在植物单细胞测序中的应用和使用方法。The invention belongs to the field of biotechnology and relates to a method for preparing plant cell nuclei, and specifically relates to a method for preparing cell nuclei of kiwifruit root tissue and an application and use method thereof in plant single-cell sequencing.
背景技术Background technique
单细胞测序技术通过测定单个细胞的转录组，以“单细胞分辨力”来解析生命现象背后的分子机制，是生命科学研究的尖端技术手段，得到了广泛应用。然而，由于细胞壁的存在，植物组织进行单细胞测序“困难重重”，必须先将植物细胞解离为原生质体才能进行单细胞测序。细胞壁的主要成分是纤维素等多糖类物质，这些大分子结构多变，化学性质稳定，是最难于降解的物质之一。因此，目前多数植物组织很难制备到满足单细胞测序要求的原生质体，极大的限制了植物单细胞测序研究。另外，制备原生质体对植物细胞造成非常大的改变，会诱发转录变化，导致测序结果可靠性严重降低，数据无法反映真实的生物学变化。Single-cell sequencing technology measures the transcriptome of a single cell and uses "single-cell resolution" to analyze the molecular mechanisms behind life phenomena. It is a cutting-edge technical means for life science research and has been widely used. However, due to the presence of cell walls, single-cell sequencing of plant tissues is "difficult". Plant cells must first be dissociated into protoplasts before single-cell sequencing can be performed. The main components of the cell wall are polysaccharides such as cellulose. These macromolecules have variable structures and stable chemical properties and are one of the most difficult substances to degrade. Therefore, it is currently difficult to prepare protoplasts that meet the requirements of single-cell sequencing from most plant tissues, which greatly limits plant single-cell sequencing research. In addition, the preparation of protoplasts causes very large changes to plant cells, which will induce transcriptional changes, resulting in a serious reduction in the reliability of sequencing results, and the data cannot reflect real biological changes.
为了规避组织分离细胞困难以及解离过程造成的数据失真等问题，动物组织会采用单细胞核测序来开展实验，收到了非常好的效果。因此，借鉴动物组织中的方式，通过制备植物细胞核来开展植物单细胞测序是突破当前技术难题的有效手段。但当前植物组织制备细胞核的方法，都是应用于拟南芥的相关研究，不适用于猕猴桃根组织研究，猕猴桃根组织制备细胞核后，细胞核数量、细胞碎片比例等指标无法满足单细胞测序的要求，亟需开发一种新的适用于猕猴桃根组织植物单细胞测序研究的细胞核制备方法。In order to circumvent the difficulties in separating cells from tissues and data distortion caused by the dissociation process, animal tissues will use single-cell nuclear sequencing to conduct experiments, which has achieved very good results. Therefore, borrowing from the methods used in animal tissues and conducting plant single-cell sequencing by preparing plant cell nuclei is an effective means to overcome current technical difficulties. However, the current methods for preparing cell nuclei from plant tissues are all applied to related research on Arabidopsis thaliana, and are not suitable for kiwifruit root tissue research. After preparing cell nuclei from kiwifruit root tissues, indicators such as the number of cell nuclei and the proportion of cell fragments cannot meet the requirements of single-cell sequencing. It is urgent to develop a new method for preparing cell nuclei suitable for single-cell sequencing research of kiwifruit root tissue plants.
发明内容Summary of the invention
拟南芥虽然有成熟的细胞核制备技术方案，如《Identification of OpenChromatin Regions in Plant Genomes Using ATAC-Seq》与《Isolation of Plant RootNuclei for Single Cell RNA Sequencing》中所述实验步骤，前者主要应用于拟南芥的基因组测序研究，后者主要应用于转录组测序研究，两种方法可以分别制备拟南芥叶和根组织的细胞核悬液。猕猴桃为大型落叶藤本，与拟南芥的组织相似性极低，细胞壁组成成分差异较大，且由于生长环境不同，组织和细胞的组成也有较大差异。使用拟南芥制备细胞核的方法无法制备满足单细胞测序的猕猴桃根组织细胞核悬液。Although Arabidopsis has mature cell nucleus preparation technology solutions, such as the experimental steps described in "Identification of OpenChromatin Regions in Plant Genomes Using ATAC-Seq" and "Isolation of Plant RootNuclei for Single Cell RNA Sequencing", the former is mainly used in Arabidopsis genome sequencing research, and the latter is mainly used in transcriptome sequencing research. The two methods can prepare cell nucleus suspensions of Arabidopsis leaf and root tissues, respectively. Kiwifruit is a large deciduous vine with very low tissue similarity to Arabidopsis, with large differences in cell wall composition, and due to different growth environments, the composition of tissues and cells is also very different. The method of preparing cell nuclei using Arabidopsis cannot prepare kiwifruit root tissue cell nucleus suspensions that meet the requirements of single-cell sequencing.
针对现有技术的空白和存在的不足，本发明在大量的深入研究和实验的基础上，提供了一种适用于单细胞测序的猕猴桃根组织细胞核的制备方法，及相应的裂解液配方和细胞核悬液优化方法(其中，本发明所述细胞核悬液优化方法，主要优化的内容为，去除猕猴桃根组织/细胞中沉积的草酸钙晶体、去除提取猕猴桃根组织过程中释放到悬液中的mRNA、去除细胞核悬液中的组织碎片)。In view of the gaps and shortcomings in the prior art, the present invention, based on a large amount of in-depth research and experiments, provides a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, as well as a corresponding lysate formula and a method for optimizing a cell nucleus suspension (wherein the cell nucleus suspension optimization method described in the present invention mainly optimizes the removal of calcium oxalate crystals deposited in kiwifruit root tissue/cells, the removal of mRNA released into the suspension during the extraction of kiwifruit root tissue, and the removal of tissue fragments in the cell nucleus suspension).
本发明提供的适用于单细胞测序的猕猴桃根组织细胞核的制备方法，包括如下具体步骤：The method for preparing kiwifruit root tissue cell nuclei suitable for single-cell sequencing provided by the present invention comprises the following specific steps:
1)配置KR裂解液：首先配置KR裂解液，并将配置好的溶液预冷。1) Prepare KR lysis buffer: First prepare KR lysis buffer and pre-cool the prepared solution.
2)破碎样本：将新鲜取材或者冻存的猕猴桃根组织样本转入离心管，加入所预冷的KR裂解液及钢珠，依照仪器操作说明置于破碎仪上对组织进行破碎。2) Sample crushing: transfer freshly collected or frozen kiwifruit root tissue samples into a centrifuge tube, add pre-cooled KR lysis solution and steel beads, and crush the tissue on a crusher according to the instrument operating instructions.
3)裂解细胞：破碎后将离心管置于冰上孵育裂解。3) Lyse cells: After disruption, place the centrifuge tube on ice and incubate for lysis.
4)筛网过滤：将步骤3)裂解后的组织裂解液转入离心管中，加入预冷的PBS。冰上静置1分钟。吸出组织裂解液，加入滤网中进行过滤。4) Screen filtration: Transfer the tissue lysate after lysis in step 3) into a centrifuge tube and add pre-cooled PBS. Let stand on ice for 1 minute. Aspirate the tissue lysate and add it to the filter for filtration.
5)筛网过滤：使用滤网对步骤4)中的过滤液再进行一次过滤。5) Screen filtration: Use a screen to filter the filtrate in step 4) again.
6)离心收集沉淀：将步骤5)中的过滤液进行离心。丢弃上清，沉淀使用PBS进行重悬。6) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 5), discard the supernatant, and resuspend the precipitate in PBS.
7)过滤：将步骤6)中的沉淀重悬液全部加入分选柱中进行过滤。7) Filtration: Add all the precipitate resuspension in step 6) into the separation column for filtration.
8)离心收集沉淀：将步骤7)中的过滤液进行离心，丢弃上清，沉淀使用100μLPBS进行重悬。8) Collecting the precipitate by centrifugation: Centrifuge the filtrate in step 7), discard the supernatant, and resuspend the precipitate in 100 μL PBS.
步骤(1)中，所述KR裂解液包含以下成份(终浓度)：In step (1), the KR lysate contains the following components (final concentration):
2％～3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15～25mM柠檬酸钠、0.2～0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2～0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)；优选地，为3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、25mM柠檬酸钠、0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613); preferably, 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 25 mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613).
步骤(1)中，L-酒石酸常用作饮料和其他食品的酸味剂，在本发明中作用为中和组织和细胞破碎后释放的细胞内容物，稳定裂解液PH，保持细胞核完整；同时L-酒石酸作为裂解液中的还原剂，发挥保持RNase inhibitor(RNA酶抑制剂)的活性的作用。In step (1), L-tartaric acid is commonly used as an acidulant for beverages and other foods. In the present invention, it serves to neutralize the cell contents released after tissue and cell disruption, stabilize the pH of the lysate, and maintain the integrity of the cell nucleus; at the same time, L-tartaric acid acts as a reducing agent in the lysate to maintain the activity of the RNase inhibitor.
步骤(1)中，皂素主要作用是医药原料，同时也被用于防腐材料、乳化剂成分，在本发明中，皂素作为裂解液的成分发挥裂解细胞膜，释放细胞核的作用。In step (1), saponin mainly functions as a pharmaceutical raw material, and is also used as a preservative material and an emulsifier component. In the present invention, saponin, as a component of a lysing solution, plays a role in lysing cell membranes and releasing cell nuclei.
步骤(1)中，配置KR裂解液时需要先加入2％～3％(v/v)L-酒石酸(NanjingReagent,C0681014023)，将溶液充分混匀并置于冰上预冷后再加入0.2～0.4U/mL RNaseinhibitor。其目的是2％～3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)可以保持0.2～0.4U/mL RNase inhibitor的活性。In step (1), when preparing the KR lysate, 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) needs to be added first, the solution is thoroughly mixed and placed on ice for precooling before adding 0.2 to 0.4 U/mL RNase inhibitor. The purpose is that 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) can maintain the activity of 0.2 to 0.4 U/mL RNase inhibitor.
步骤(1)中，所述预冷温度为0～5℃；优选地，为放置于4℃冰箱预冷。In step (1), the precooling temperature is 0-5°C; preferably, it is precooled in a 4°C refrigerator.
步骤(2)中，所述离心管为 2.0mL MaxyClear SnaplockMicrocentrifuge Tube，RNase-/DNase-free and nonpyrogenic(Axygen,MCT-200-C)(爱思进2.0ml无核酸酶透明离心管)。In step (2), the centrifuge tube is a 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, RNase-/DNase-free and nonpyrogenic (Axygen, MCT-200-C).
步骤(2)中，破碎仪常用于核酸抽提工作，在本发明中的作用是通过钢珠充分破坏植物组织，辅助KR裂解液完成粗细胞核悬液的制备。步骤(2)中，所述钢珠的直径为5mm。步骤(2)中，所述破碎仪为万柏生物高通量组织破碎仪(onebio-48p)。步骤(2)中，所述破碎的条件为1200～1300rpm，170～180s；优选地，为1200rpm，180s。步骤(2)中，所述猕猴桃根可指任意品种猕猴桃的根尖组织，包括分生区、伸长区、成熟区。步骤(2)中，所述猕猴桃根组织，其鲜重为50～100mg；优选地，为100mg。In step (2), the crusher is commonly used in nucleic acid extraction. Its role in the present invention is to fully destroy plant tissues through steel balls, and assist KR lysis solution to complete the preparation of crude cell nucleus suspension. In step (2), the diameter of the steel balls is 5 mm. In step (2), the crusher is Wanbo Bio high-throughput tissue crusher (onebio-48p). In step (2), the crushing conditions are 1200-1300 rpm, 170-180 s; preferably, 1200 rpm, 180 s. In step (2), the kiwifruit root may refer to the root tip tissue of any variety of kiwifruit, including meristem, elongation zone, and ripening zone. In step (2), the fresh weight of the kiwifruit root tissue is 50-100 mg; preferably, 100 mg.
步骤(3)中，所述孵育的目的是在保持细胞核内信使RNA(mRNA)完整性的温度下，使用KR裂解液去除细胞核外的细胞膜等结构。步骤(3)中，所述孵育的时间为7～8分钟；优选地，为7min。In step (3), the purpose of the incubation is to use KR lysis solution to remove the cell membrane and other structures outside the cell nucleus at a temperature that maintains the integrity of the messenger RNA (mRNA) in the cell nucleus. In step (3), the incubation time is 7 to 8 minutes; preferably, 7 minutes.
步骤(4)中，所述滤网为FALCON 40μm Cell Strainer(FALCON,352340)，孔径40μm。步骤(4)中，所述PBS为Gibco PBS pH7.4(1×)(Gibco,10010-031)。步骤(4)中，所述离心管为Corning Centristar 15mL离心管(Corning,430790)。步骤(4)中，所述过滤全程在0～5℃操作；优选地，为放置冰上操作。步骤(4)中冰上静置1分钟的目的是使大的组织块沉降至离心管底部，静置时间过短组织块沉降不完全，造成筛网过滤时筛网堵塞，导致细胞核损失；静置时间过长则存在mRNA降解的风险，mRNA的降解将直接导致实验的失败。组织块沉降的时间为1min可以确保沉降完全且无mRNA降解的风险。In step (4), the filter is FALCON 40μm Cell Strainer (FALCON, 352340) with a pore size of 40μm. In step (4), the PBS is Gibco PBS pH7.4 (1×) (Gibco, 10010-031). In step (4), the centrifuge tube is Corning Centristar 15mL centrifuge tube (Corning, 430790). In step (4), the filtration is performed at 0-5°C throughout the entire process; preferably, the operation is performed on ice. The purpose of standing on ice for 1 minute in step (4) is to allow large tissue blocks to settle to the bottom of the centrifuge tube. If the standing time is too short, the tissue blocks will not settle completely, causing the screen to be blocked during filtration, resulting in loss of cell nuclei; if the standing time is too long, there is a risk of mRNA degradation, and the degradation of mRNA will directly lead to the failure of the experiment. The tissue block sedimentation time of 1min can ensure complete sedimentation without the risk of mRNA degradation.
步骤(5)中，所述滤网为FALCON 40μm Cell Strainer(FALCON,352340)，孔径40μm。In step (5), the filter is FALCON 40 μm Cell Strainer (FALCON, 352340) with a pore size of 40 μm.
步骤(6)中，所述离心的温度为4-6℃；优选地，为4℃。步骤(6)中，所述离心的时间为10-15分钟；优选地，为10分钟。步骤(6)中，所述离心的离心力为300～500×g，优选地，为300×g。步骤(6)中，所述PBS为提前30min置于4℃冰箱中预冷后的PBS。In step (6), the centrifugation temperature is 4-6°C; preferably, 4°C. In step (6), the centrifugation time is 10-15 minutes; preferably, 10 minutes. In step (6), the centrifugal force of the centrifugation is 300-500×g, preferably, 300×g. In step (6), the PBS is PBS that has been precooled in a 4°C refrigerator for 30 minutes in advance.
步骤(7)中，所述分选柱为Miltenyi MS Column(Miltenyi,130-042-201)。步骤(7)中，所述分选柱通常用于磁场下，通过带有磁珠的抗体结合目的细胞膜表面的特异性抗原，从而用于动物特定细胞类型的分选，在本发明中，在非磁场下，利用草酸钙晶体的针状形态特征和分选柱中纳米材料的堆叠的特点，体积小的细胞核可以伴随液流穿过分选柱，针状草酸钙晶体则会被留在分选柱中，无法伴随液流穿过，达到纯化细胞核的目的。步骤(7)中，所述过滤全程在0～5℃操作；优选地，为放置冰上操作。In step (7), the sorting column is a Miltenyi MS Column (Miltenyi, 130-042-201). In step (7), the sorting column is usually used in a magnetic field to bind specific antigens on the surface of the target cell membrane through antibodies with magnetic beads, so as to be used for sorting specific cell types of animals. In the present invention, in a non-magnetic field, the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized, and small cell nuclei can pass through the sorting column with the liquid flow, while the needle-shaped calcium oxalate crystals will be retained in the sorting column and cannot pass through with the liquid flow, thereby achieving the purpose of purifying cell nuclei. In step (7), the filtration is operated at 0-5°C throughout the whole process; preferably, it is placed on ice for operation.
步骤(8)中，所述离心的温度为4-6℃；优选地，为4℃。步骤(8)中，所述离心的时间为10-15分钟；优选地，为10分钟。步骤(8)中，所述离心的离心力为300～500×g；优选地，为300×g。步骤(8)中，所述PBS为冰上预冷后的PBS。In step (8), the centrifugation temperature is 4-6°C; preferably, 4°C. In step (8), the centrifugation time is 10-15 minutes; preferably, 10 minutes. In step (8), the centrifugal force is 300-500×g; preferably, 300×g. In step (8), the PBS is PBS pre-cooled on ice.
本发明还提供了由上述方法得到的适用于单细胞测序的猕猴桃根组织细胞核。The present invention also provides kiwifruit root tissue cell nuclei suitable for single-cell sequencing obtained by the above method.
本发明还提供了所述猕猴桃根组织细胞核在单细胞测序中的应用。The present invention also provides the application of the kiwifruit root tissue cell nucleus in single-cell sequencing.
在一个具体的实施方式中，本发明所述方法的具体步骤为：In a specific embodiment, the specific steps of the method of the present invention are:
1)配置KR裂解液。1) Prepare KR lysis buffer.
依照以下成分配置裂解液： 2％～3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15～25mM柠檬酸钠、0.2～0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2～0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。The lysis buffer was prepared according to the following ingredients: 2%-3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.2-0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613).
将配置好的裂解液置于冰上进行预冷。Place the prepared lysate on ice for pre-cooling.
2)破碎样本。将新鲜取材或者冻存的猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200～1300rpm，170～180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer fresh or frozen kiwi root tissue samples into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200-1300 rpm for 170-180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate one by one and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828).
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。5) Filtration. Use a pipette to gradually aspirate the filtrate in step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C.
离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 100 μL of ice-cold PBS.
9)镜检计数。取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀。使用血球计数板计数细胞核总量和浓度。9) Microscopic examination and counting. Take 5 μL of the resuspension in step (8) and mix it with DAPI staining solution in a ratio of 1:1. Use a hemocytometer to count the total number and concentration of cell nuclei.
10)单细胞测序上机。依照10x Genomics试剂盒说明书操作，上机进行单细胞测序。10) Single-cell sequencing: Follow the instructions of the 10x Genomics kit and perform single-cell sequencing.
本发明还提供了一种KR裂解液，所述KR裂解液包含以下成份(终浓度)：2％～3％(v/v)L-酒石酸、15～25mM柠檬酸钠、0.2～0.4％(m/v)皂素、0.2～0.4U/mL重组型RNase抑制剂。The present invention also provides a KR lysate, which comprises the following components (final concentration): 2% to 3% (v/v) L-tartaric acid, 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin, and 0.2 to 0.4 U/mL recombinant RNase inhibitor.
本发明还提供了一种试剂/试剂盒，其特征在于，其包含如上所述的KR裂解液。The present invention also provides a reagent/kit, characterized in that it comprises the KR lysate as described above.
本发明还提供了所述的KR裂解液或所述的试剂/试剂盒在制备适用于单细胞测序的猕猴桃根组织细胞核的方法、猕猴桃根组织细胞核转录组测序、猕猴桃根组织细胞核ATAC测序研究中的应用。The present invention also provides the use of the KR lysate or the reagent/kit in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, kiwifruit root tissue nuclei transcriptome sequencing, and kiwifruit root tissue nuclei ATAC sequencing research.
与现有技术相比，本发明取得的有益效果如下：Compared with the prior art, the present invention has the following beneficial effects:
第一，填补了目前的技术空白：目前缺少适用于单细胞测序实验的猕猴桃根组织细胞核的制备方法，严重阻碍了单细胞测序技术在猕猴桃根组织研究中的应用。本发明提供的技术方案解决了这一问题，将有力的促进猕猴桃根组织单细胞测序研究的开展和广泛应用。First, it fills the current technical gap: the lack of a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing experiments has seriously hindered the application of single-cell sequencing technology in kiwifruit root tissue research. The technical solution provided by the present invention solves this problem and will effectively promote the development and widespread application of kiwifruit root tissue single-cell sequencing research.
第二，猕猴桃根细胞核悬液质量提升。猕猴桃根组织中含有大量杂质、多糖等，裂解后的悬液中存在大量碎片、细胞内晶体等杂质，其质量完全无法满足10x Genomics上机要求，严重阻碍了猕猴桃根组织的研究进程。而根据本发明技术方案制备得到的细胞核质量很高(样本质量从细胞核数量3×105个，碎片率大于70％，提升到2.26×106～4.12×106个，碎片率3～5％。)，细胞碎片、细胞内晶体等杂质基本被完全去除，可以顺利的开展后续的单细胞实验。Second, the quality of kiwifruit root nucleus suspension is improved. Kiwifruit root tissue contains a large amount of impurities, polysaccharides, etc., and there are a large number of debris, intracellular crystals and other impurities in the suspension after lysis. Its quality is completely unable to meet the requirements of 10x Genomics, which seriously hinders the research progress of kiwifruit root tissue. The quality of the nuclei prepared according to the technical solution of the present invention is very high (the sample quality is improved from 3×105 nuclei and a fragmentation rate of more than 70% to 2.26×106~4.12×106 and a fragmentation rate of 3~5%.), cell fragments, intracellular crystals and other impurities are basically completely removed, and subsequent single-cell experiments can be carried out smoothly.
第三，成本低廉，耗时较短。本发明提供的技术方案使用的裂解液组分仅有4种，与现有技术中6～7种组分相比有着显著的减少；实验流程在30分钟内即可完成细胞核的制备。避免了蔗糖沉积等步骤，节约了试剂、耗材，降低了成本，同时大大缩短了时间，提高了效率，便于大量样本实验的开展。Third, it is low-cost and time-consuming. The technical solution provided by the present invention uses only 4 lysate components, which is significantly reduced compared with 6 to 7 components in the prior art; the experimental process can complete the preparation of cell nuclei within 30 minutes. It avoids steps such as sucrose deposition, saves reagents and consumables, reduces costs, and greatly shortens time, improves efficiency, and facilitates the development of large-scale sample experiments.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是细胞核镜检图。图示为经本发明技术方法制备得到的细胞核。图中可见细胞碎片、草酸钙晶体等杂质被完全去除，仅存在占比很低的小于细胞核的杂质。Figure 1 is a microscopic examination of a cell nucleus. The figure shows a cell nucleus prepared by the technical method of the present invention. It can be seen in the figure that impurities such as cell debris and calcium oxalate crystals are completely removed, and only a very small proportion of impurities smaller than the cell nucleus exist.
图2是本发明实施例1～4单细胞转录组测序cDNA质检结果(安捷伦4150生物分析仪)。FIG. 2 is the cDNA quality inspection results of single-cell transcriptome sequencing of Examples 1 to 4 of the present invention (Agilent 4150 Bioanalyzer).
图3是组织中草酸钙针状晶体图示。FIG. 3 is a diagram of needle-shaped calcium oxalate crystals in tissue.
实施方式Implementation
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
实施例1Example 1
1)配置裂解液。依照以下成分配置裂解液： 2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用的夹板并拧紧扳手，调节仪器转速为1200rpm，170秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 170 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
9)洗涤沉淀；将步骤(8)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。9) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
10)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量悬液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(9)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。10) Quality inspection, microscopic examination and counting. Take the supernatant and use NanoDrop micro-spectrophotometer and fluorescence spectrophotometer to measure the concentration of RNA in the suspension. Use DAPI staining solution and calculate the concentration of live cell nuclei, agglomeration ratio, and impurity ratio under a microscope: take 5 μL of the resuspension in step (9), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to take 10 ul and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
11)单细胞测序上机。依照10x Genomics公司说明书：ChromiumNext GEM Single Cell 3’Reagent Kitv3.1，CG000204 REV C，进行单细胞测序操作。11) Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3’Reagent Kit v3.1, CG000204 REV C.
结果及分析：结果见表1；活细胞比例、细胞结团比例、碎片比例和细胞浓度均能达到10x Genomics公司单细胞测序要求(表1，图1)、cDNA长度正常(图2)。文库构建结果显示冻存样本提取细胞核悬液进行单细胞转录组测序效果良好。Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal (Figure 2). The library construction results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has a good effect.
实施例2Example 2
1)配置裂解液。依照以下成分配置裂解液： 3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、25mM柠檬酸钠、0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1300rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1300 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育8分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for incubation for 8 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
9)洗涤沉淀；将步骤(8)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。9) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
10)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量悬液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(9)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。10) Quality inspection, microscopic examination and counting. Take the supernatant and use NanoDrop micro-spectrophotometer and fluorescence spectrophotometer to measure the concentration of RNA in the suspension. Use DAPI staining solution and calculate the concentration of live cell nuclei, agglomeration ratio, and impurity ratio under a microscope: take 5 μL of the resuspension in step (9), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to take 10 ul and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
11)单细胞测序上机。依照10x Genomics公司说明书：ChromiumNext GEM Single Cell 3’Reagent Kitv3.1，CG000204REV C，进行单细胞测序操作。11) Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3’Reagent Kit v3.1, CG000204REV C.
结果及分析：结果见表1；悬液中RNA含量极低；活细胞比例、细胞结团比例、碎片比例和细胞浓度均能达到10x Genomics公司单细胞测序要求、cDNA长度正常，文库构建结果显示冻存样本提取细胞核悬液进行单细胞转录组测序效果良好(图2)。Results and analysis: The results are shown in Table 1. The RNA content in the suspension was extremely low. The proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all met the single-cell sequencing requirements of 10x Genomics. The cDNA length was normal. The library construction results showed that the single-cell transcriptome sequencing of the cell nucleus suspension extracted from the frozen samples was effective (Figure 2).
实施例3Example 3
1)配置裂解液。依照以下成分配置裂解液： 2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，170秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 170 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
9)洗涤沉淀；将步骤(8)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。9) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
10)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量悬液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(9)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。10) Quality inspection, microscopic examination and counting. Take the supernatant and use a NanoDrop micro-spectrophotometer and a fluorescence spectrophotometer to measure the RNA concentration in the suspension. Use DAPI staining solution and calculate the concentration of live cell nuclei, agglomeration ratio, and impurity ratio under a microscope: take 5 μL of the resuspension in step (9), mix it with DAPI staining solution in a 1:1 ratio, use a pipette to take 10 ul and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
11)单细胞测序上机。依照10x Genomics公司说明书：ChromiumNext GEM Single Cell 3’Reagent Kitv3.1，CG000204REV C，进行单细胞测序操作。11) Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3’Reagent Kit v3.1, CG000204REV C.
结果及分析：结果见表1；活细胞比例、细胞结团比例、碎片比例和细胞浓度均能达到10x Genomics公司单细胞测序要求(表1，图1)、cDNA长度正常(图2)。文库构建结果显示冻存样本提取细胞核悬液进行单细胞转录组测序效果良好。Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal (Figure 2). The library construction results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has good results.
实施例4Example 4
1)配置裂解液。依照以下成分配置裂解液： 3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、25mM柠檬酸钠、0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将100mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1300rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 100 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1300 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育8分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for incubation for 8 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
9)洗涤沉淀；将步骤(8)中的离心管置于离心机中，4℃下，500×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。9) Wash the precipitate; place the centrifuge tube in step (8) in a centrifuge and centrifuge at 500×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
10)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量悬液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(9)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。10) Quality inspection, microscopic examination and counting. Take the supernatant and use NanoDrop micro-spectrophotometer and fluorescence spectrophotometer to measure the concentration of RNA in the suspension. Use DAPI staining solution and calculate the concentration of live cell nuclei, agglomeration ratio, and impurity ratio under a microscope: take 5 μL of the resuspension in step (9), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to take 10 ul and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
11)单细胞测序上机。依照10x Genomics公司说明书：ChromiumNext GEM Single Cell 3’Reagent Kitv3.1，CG000204 REV C，进行单细胞测序操作。11) Single-cell sequencing was performed according to the instructions of 10x Genomics: ChromiumNext GEM Single Cell 3’Reagent Kit v3.1, CG000204 REV C.
结果及分析：结果见表1；活细胞比例、细胞结团比例、碎片比例和细胞浓度均能达到10x Genomics公司单细胞测序要求(表1，图1)、cDNA长度正常(图2)。文库构建结果显示冻存样本提取细胞核悬液进行单细胞转录组测序效果良好。Results and analysis: The results are shown in Table 1; the proportion of live cells, cell agglomeration ratio, fragment ratio and cell concentration all meet the requirements of 10x Genomics single-cell sequencing (Table 1, Figure 1), and the cDNA length is normal (Figure 2). The library construction results show that the single-cell transcriptome sequencing of cell nucleus suspension extracted from frozen samples has good results.
对比实施例1Comparative Example 1
本实施例依照《Identification of Open Chromatin Regions in PlantGenomes Using ATAC-Seq》进行拟南芥叶片细胞核制备。In this example, Arabidopsis leaf nuclei were prepared according to "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq".
1)配置裂解液。依照以下成分配置细胞核纯化缓冲液20mM MOPS pH 7,40mM NaCl,90mM KCl,2mM EDTA,0.5mM EGTA,0.5mMspermidine,0.2mM spermine,and 1×Roche Complete protease inhibitors。依照以下成分配置细胞核提取液1： 0.25M Sucrose,10mM Tris–HCl pH 8,10mM MgCl2,1％Triton X-100,and 1×Roche Complete Protease Inhibitors。依照以下成分配置细胞核提取液2： 1.7M Sucrose,10mM Tris–HCl pH 8,2mM MgCl2,and 0.15％Triton X-100,1×Roche Complete Protease Inhibitors。将配置好的溶液置于冰上进行预冷。1) Prepare lysis buffer. Prepare nuclear purification buffer according to the following ingredients: 20mM MOPS pH 7, 40mM NaCl, 90mM KCl, 2mM EDTA, 0.5mM EGTA, 0.5mM spermine, 0.2mM spermine, and 1×Roche Complete protease inhibitors. Prepare nuclear extraction buffer 1 according to the following ingredients: 0.25M Sucrose, 10mM Tris–HCl pH 8, 10mM MgCl2, 1% Triton X-100, and 1×Roche Complete Protease Inhibitors. Prepare nuclear extraction buffer 2 according to the following ingredients: 1.7M Sucrose, 10mM Tris–HCl pH 8, 2mM MgCl2, and 0.15% Triton X-100, 1×Roche Complete Protease Inhibitors. Pre-cool the prepared solutions on ice.
2)破碎样本。将50mg拟南芥叶片组织样本转入研钵中，倒入一定量液氮进行研磨，至组织全部变为粉末为止。期间注意添加液氮，以保持低温。2) Crush the sample. Place 50 mg of Arabidopsis leaf tissue sample into a mortar and add a certain amount of liquid nitrogen to grind until the tissue is completely powdered. During this period, be sure to add liquid nitrogen to keep the temperature low.
3)裂解细胞。将研磨的粉末转入一个新的含有10mL冰上预冷的细胞核纯化缓冲液的研钵中。使用研棒将粉末搅拌重悬。3) Lyse the cells. Transfer the ground powder into a new mortar containing 10 mL of ice-cold nuclear purification buffer. Use a pestle to stir and resuspend the powder.
4)过滤。用移液器将步骤(3)中的组织裂解液逐次吸出，于70μm滤网进行过滤，滤液收集到一个新的15mL离心管中。注意离心管应插于冰中。4) Filtration. Use a pipette to gradually aspirate the tissue lysate in step (3), filter it through a 70 μm filter, and collect the filtrate into a new 15 mL centrifuge tube. Note that the centrifuge tube should be placed in ice.
5)离心收集沉淀。将步骤(4)中的离心管，于离心机中以1200×g、4℃离心10分钟。小心的取出离心管，将上清吸出弃去。5) Collect the precipitate by centrifugation. Centrifuge the centrifuge tube in step (4) at 1200×g and 4°C for 10 minutes. Carefully remove the centrifuge tube and aspirate and discard the supernatant.
6)离心收集沉淀。使用1mL预冷的细胞核提取液1将步骤(5)中的细胞核沉淀重悬。将重悬液吸出，加入一个新的1.5mL离心管中。将离心管置于离心机中，以12000×g、4℃离心10分钟。小心的取出离心管，将上清吸出弃去。6) Collect the precipitate by centrifugation. Resuspend the nuclear precipitate in step (5) with 1 mL of pre-cooled nuclear extraction solution 1. Aspirate the resuspended solution and add it to a new 1.5 mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 × g and 4°C for 10 minutes. Carefully remove the centrifuge tube and aspirate and discard the supernatant.
7)离心收集沉淀。向一个新的1.5mL离心管中加入300μL细胞核提取液2。使用300μL预冷的细胞核提取液2将步骤(6)中的细胞核沉淀重悬。将重悬液小心的加入到含有细胞核提取液2的离心管中，使溶液分层。将离心管置于离心机中，以16000×g、4℃离心10分钟。小心的取出离心管，将上清吸出弃去。使用1mL预冷的细胞核纯化缓冲液重悬沉淀。7) Collect the precipitate by centrifugation. Add 300 μL of nuclear extract solution 2 to a new 1.5 mL centrifuge tube. Resuspend the nuclear precipitate from step (6) with 300 μL of pre-cooled nuclear extract solution 2. Carefully add the resuspension to the centrifuge tube containing nuclear extract solution 2 to separate the solutions. Place the centrifuge tube in a centrifuge and centrifuge at 16,000 × g and 4°C for 10 minutes. Carefully remove the centrifuge tube and aspirate and discard the supernatant. Resuspend the precipitate with 1 mL of pre-cooled nuclear purification buffer.
8)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。8) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 μL of the resuspension in step (8), mix it with DAPI stain in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
9)结果及分析；结果显示细胞核数量为2×106个，碎片杂质占比28％。满足单细胞转录组测序对于样本的最低要求。9) Results and analysis: The results showed that the number of cell nuclei was 2×106, and the proportion of debris impurities was 28%, which met the minimum sample requirements for single-cell transcriptome sequencing.
对比实施例2Comparative Example 2
本实施例依照《Identification of Open Chromatin Regions in PlantGenomes Using ATAC-Seq》进行猕猴桃叶片组织细胞核制备。其余操作步骤与对比实施例1相同。This example is based on "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" to prepare kiwifruit leaf tissue nuclei. The remaining steps are the same as those in Comparative Example 1.
结果见表2。The results are shown in Table 2.
对比实施例3Comparative Example 3
本实施例依照《Identification of Open Chromatin Regions in PlantGenomes Using ATAC-Seq》进行猕猴桃根组织细胞核制备。其余操作步骤与对比实施例1相同。This example is based on "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" to prepare kiwifruit root tissue nuclei. The remaining steps are the same as those in Comparative Example 1.
结果见表2。The results are shown in Table 2.
对比实施例4Comparative Example 4
本实施例依照依照《Isolation of Plant Root Nuclei for Single Cell RNASequencing》中植物组织细胞核提取试剂盒(CelLyticTM PN分离/提取试剂盒，sigma，编号为CELLYTPN1-1KT)进行拟南芥根组织细胞核制备。In this example, Arabidopsis root tissue nuclei were prepared according to the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing".
1)准备裂解液；将500μLNIB裂解液倒入新的、无菌且预冷藏的60mm培养皿中。1) Prepare lysis buffer: Pour 500 μL of NIB lysis buffer into a new, sterile and pre-chilled 60 mm culture dish.
2)取材；将7日龄的拟南芥植物的根部用双面刀片切下，收集新鲜根部。2) Sampling: The roots of 7-day-old Arabidopsis plants were cut with a double-edged blade to collect fresh roots.
3)转移材料；用镊子将新鲜根部样品转移到60mm培养皿中，并将根全部浸入NIB裂解液中。3) Transferring materials: Use forceps to transfer fresh root samples to a 60 mm culture dish and immerse all roots in NIB lysis solution.
4)组织切碎；将取到的根部样本用双面刀片切碎约5分钟。4) Tissue mincing: mince the root sample using a double-sided blade for about 5 minutes.
5)组织裂解；在4℃环境中轻轻水平摇动孵育15分钟进行裂解。5) Tissue lysis: Incubate at 4°C with gentle horizontal shaking for 15 minutes to allow lysis.
6)过滤；在低温环境下，先使用500μLNIB将40μm过滤器润湿，随后用一次性大口径吸头吸取根裂解后的核悬液，缓慢进行过滤，再使用500μLNIB对过滤器进行冲洗，收集剩余细胞核。6) Filtration; Under low temperature, first use 500 μL NIB to wet the 40 μm filter, then use a disposable large-bore pipette tip to absorb the nuclear suspension after root lysis, slowly filter, and then use 500 μL NIB to rinse the filter and collect the remaining cell nuclei.
7)筛网过滤；再将过滤后的核悬液用同步骤7的方法，使用30μm过滤器进行二次过滤，再使用500μLNIB对过滤器进行冲洗，收集剩余细胞核，并去除碎片。7) Filter through a mesh; filter the filtered nuclear suspension for a second time using a 30 μm filter in the same manner as in step 7, and then rinse the filter with 500 μL of NIB to collect the remaining nuclei and remove the debris.
8)镜检计数；通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10uL加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。8) Microscopic examination and counting; Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope by using DAPI staining solution: Take 5 μL of the resuspension in step (8), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
结果见表2The results are shown in Table 2
对比实施例5Comparative Example 5
本实施例依照依照《Isolation of Plant Root Nuclei for Single Cell RNASequencing》中植物组织细胞核提取试剂盒(CelLyticTM PN分离/提取试剂盒，sigma，编号为CELLYTPN1-1KT)进行猕猴桃叶片组织细胞核制备。其余操作步骤与对比实施例4相同。In this example, the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" was used to prepare the kiwifruit leaf tissue nucleus. The remaining steps were the same as those in Comparative Example 4.
结果见表2。The results are shown in Table 2.
对比实施例6Comparative Example 6
本实施例依照依照《Isolation of Plant Root Nuclei for Single Cell RNASequencing》中植物组织细胞核提取试剂盒(CelLyticTM PN分离/提取试剂盒，sigma，编号为CELLYTPN1-1KT)进行猕猴桃根组织细胞核制备。其余操作步骤与对比实施例4相同。This example uses the plant tissue nucleus extraction kit (CelLyticTM PN isolation/extraction kit, Sigma, numbered CELLYTPN1-1KT) in "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" to prepare kiwifruit root tissue nuclei. The remaining steps are the same as those in Comparative Example 4.
结果见表2。The results are shown in Table 2.
对比实施例7Comparative Example 7
本实施例使用ST1缓冲液进行猕猴桃根组织细胞核制备。In this example, ST1 buffer was used to prepare kiwifruit root tissue cell nuclei.
1)配置ST1缓冲液。依照以下成分配置缓冲液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)。将配置好的缓冲液置于冰上进行预冷。1) Prepare ST1 buffer. Prepare the buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023). Pre-cool the prepared buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入研钵中，倒入一定量液氮进行研磨，至组织全部变为粉末为止。期间注意添加液氮，以保持低温。2) Crush the sample. Put 50 mg of kiwi root tissue sample into a mortar and add a certain amount of liquid nitrogen to grind until the tissue is completely powdered. During this period, be sure to add liquid nitrogen to keep the temperature low.
3)裂解细胞。将研磨的粉末转入一个新的含有10mL冰上预冷的缓冲液的15mL离心管中。上下颠倒15次重悬。3) Lyse cells. Transfer the ground powder into a new 15 mL centrifuge tube containing 10 mL of ice-cold buffer. Invert 15 times to resuspend.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的细胞核悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 μL of the cell nucleus suspension in step (3), mix it with DAPI stain in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
5)参数测量。使用pH计(METTLER TOLEDO FE28-CN，30595013)测量步骤(3)中缓冲液pH。5) Parameter measurement: The pH of the buffer solution in step (3) was measured using a pH meter (METTLER TOLEDO FE28-CN, 30595013).
结果见表2。The results are shown in Table 2.
对比实施例8Comparative Example 8
本实施例使用ST2缓冲液成分为15mM柠檬酸钠进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例7相同。结果见表2。In this example, ST2 buffer containing 15 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining steps were the same as those in Comparative Example 7. The results are shown in Table 2.
对比实施例9Comparative Example 9
本实施例使用ST3缓冲液成分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、20mM Tris-HCl(pH＝7.5)进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例7相同。In this example, ST3 buffer containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 20 mM Tris-HCl (pH=7.5) was used to prepare kiwifruit root tissue nuclei, and the remaining operating steps were the same as those in Comparative Example 7.
对比实施例10Comparative Example 10
本实施例使用ST4缓冲液成分为15mM柠檬酸钠,20Mm MOPS(3-(N-吗啡啉)丙磺酸,sigma-Aldrich，1132-61-2)进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例7相同。结果见表2。In this example, ST4 buffer containing 15 mM sodium citrate and 20 mM MOPS (3-(N-morpholine) propanesulfonic acid, Sigma-Aldrich, 1132-61-2) was used to prepare kiwifruit root tissue nuclei, and the remaining steps were the same as those in Comparative Example 7. The results are shown in Table 2.
对比实施例11Comparative Example 11
本实施例使用ST缓冲液成分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例7相同。结果见表2。In this example, ST buffer containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining steps were the same as those in Comparative Example 7. The results are shown in Table 2.
对比实施例12Comparative Example 12
本实施例使用ST5缓冲液成分为1.5％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、10mM柠檬酸钠进行猕猴桃根组织细胞核制备，其余操作与对比实施例7相同。结果见表2。In this example, ST5 buffer containing 1.5% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 10 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining operations were the same as those in Comparative Example 7. The results are shown in Table 2.
对比实施例13Comparative Example 13
本实施例使用ST6缓冲液成分为4％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、30mM柠檬酸钠进行猕猴桃根组织细胞核制备，其余操作与对比实施例7相同。结果见表2。In this example, ST6 buffer containing 4% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 30 mM sodium citrate was used to prepare kiwifruit root tissue nuclei, and the remaining operations were the same as those in Comparative Example 7. The results are shown in Table 2.
对比实施例14Comparative Example 14
本实施例依照使用KR1裂解液进行猕猴桃根组织细胞核制备。This example uses KR1 lysis buffer to prepare kiwifruit root tissue nuclei.
1)配置KR1裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)SDS(十二烷基硫酸钠，Sigma,151-21-3)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR1 lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) SDS (sodium dodecyl sulfate, Sigma, 151-21-3). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，在裂解液中使用灭菌剪刀将猕猴桃根剪碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1) and chop the kiwifruit root into pieces using sterilized scissors in the lysis solution.
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表2。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 2.
对比实施例15Comparative Example 15
本实施例依照使用KR2裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)脱氧胆酸钠(Sigma,302-95-4)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nucleus was prepared by using KR2 lysate with components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) sodium deoxycholate (Sigma, 302-95-4), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例16Comparative Example 16
本实施例依照使用KR3裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)洋地黄皂苷(Sigma,11024-24-1)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nuclei were prepared by using KR3 lysate with components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) digitonin (Sigma, 11024-24-1), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例17Comparative Example 17
本实施例依照使用KR4裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(v/v)Triton X-100(4-(1,1,3,3-四甲基丁基)苯基-聚乙二醇，Sigma,9036-19-5)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nucleus was prepared by using KR4 lysate components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (v/v) Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, Sigma, 9036-19-5), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例18Comparative Example 18
本实施例依照使用KR5裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nuclei were prepared by using KR5 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例19Comparative Example 19
本实施例依照使用KR6裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.1％(m/v)皂素(Nanjing Reagent,8047-15-2)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nuclei were prepared by using KR6 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.1% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例20Comparative Example 20
本实施例依照使用KR7裂解液组分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)，进行猕猴桃根组织细胞核制备，其余操作步骤与对比实施例14相同。结果见表2。In this example, the kiwifruit root tissue nuclei were prepared by using KR7 lysate containing 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, and 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the remaining steps were the same as those in Comparative Example 14. The results are shown in Table 2.
对比实施例21Comparative Example 21
本实施例依照使用双面刀片切碎猕猴桃根组织，KR5裂解液裂解。This example uses a double-sided blade to chop kiwifruit root tissue and KR5 lysis buffer for lysis.
1)配置KR5裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR5 lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2). Place the prepared lysis buffer on ice for precooling.
2)破碎样本。将50mg猕猴桃根组织样本放置于冰上的培养皿中，使用双面刀片将根切为0.5mm的小段，转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL。2) Disintegrate the sample. Place 50 mg of kiwifruit root tissue sample in a petri dish on ice, cut the root into 0.5 mm segments using a double-sided blade, and transfer to a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1).
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表3。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 3.
对比实施例22Comparative Example 22
本实施例依照使用剪刀剪碎猕猴桃根组织，KR5裂解液裂解，操作与对比实施例17相同。结果见表3。In this example, kiwifruit root tissue was cut into pieces using scissors and lysed using KR5 lysis solution, and the operation was the same as that of comparative example 17. The results are shown in Table 3.
对比实施例23Comparative Example 23
本实施例依照使用玻璃匀浆器匀浆破碎猕猴桃根组织，KR5裂解液裂解。In this example, kiwifruit root tissue was homogenized and broken using a glass homogenizer, and lysed using KR5 lysis buffer.
1)配置KR4裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本，转入1mL玻璃匀浆器(Solarbio,YA0850)中，加入步骤(1)中冰上预冷的裂解液1mL，使用玻璃匀浆器捣20次破碎组织。将匀浆器中的液体转移至2mL离心管(Axygen,MCT-200-C)中。2) Disintegrate the sample. Transfer 50 mg of kiwifruit root tissue sample into a 1 mL glass homogenizer (Solarbio, YA0850), add 1 mL of the lysis solution pre-cooled on ice in step (1), and use the glass homogenizer to pound 20 times to disintegrate the tissue. Transfer the liquid in the homogenizer to a 2 mL centrifuge tube (Axygen, MCT-200-C).
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表3。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 3.
对比实施例24Comparative Example 24
本实施例依照使用破碎仪破碎猕猴桃根组织，KR5裂解液裂解。In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR5 lysis buffer.
1)配置KR4裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本，转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL。快速组织低温破碎匀浆仪(上海净信，JXFSTPRP-I-02)。加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Rapid tissue low-temperature crushing homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Add steel balls for crushing. Place the centrifuge tube on the crushing splint and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表3。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 3.
对比实施例25Comparative Example 25
本实施例依照使用液氮研磨破碎猕猴桃根组织，KR5裂解液裂解。This example uses liquid nitrogen to grind and crush kiwifruit root tissue, and KR5 lysis buffer for lysis.
1)配置KR4裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR4 lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入研钵中，倒入一定量液氮进行研磨，至组织全部变为粉末为止。期间注意添加液氮，以保持低温，将研磨的粉末移至2mL离心管(Axygen,MCT-200-C)中，加入步骤(1)中冰上预冷的裂解液1mL。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a mortar and add a certain amount of liquid nitrogen to grind until the tissue is completely powdered. During this period, be sure to add liquid nitrogen to maintain a low temperature. Transfer the ground powder to a 2 mL centrifuge tube (Axygen, MCT-200-C) and add 1 mL of the lysis solution pre-cooled on ice in step (1).
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表3。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 3.
对比实施例26Comparative Example 26
本实施例依照使用破碎仪破碎猕猴桃根组织，KR8裂解液裂解。In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR8 lysis buffer.
1)配置KR8裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.1U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR8 lysis buffer. Prepare the lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.1U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本，转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL。万柏生物高通量组织研磨仪(onebio-48p)。加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Onebio high-throughput tissue grinder (onebio-48p). Add steel beads for crushing. Place the centrifuge tube on the crushing splint and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope by using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
5)RNA质检。使用植物总RNA提取试剂盒(天根生化科技有限公司，DP432)抽提步骤3中细胞核总RNA。使用Agilent 2100生物分析仪质检RNA。结果见表45) RNA quality inspection. The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). RNA quality inspection was performed using an Agilent 2100 bioanalyzer. The results are shown in Table 4
对比实施例27Comparative Example 27
本实施例依照使用破碎仪破碎猕猴桃根组织，KR9裂解液裂解。In this example, kiwifruit root tissue was broken using a disruptor and lysed using KR9 lysis buffer.
1)配置KR9裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare KR9 lysis buffer. Prepare the lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本，转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL。快速组织低温破碎匀浆仪(上海净信，JXFSTPRP-I-02)。加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwi root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of lysis solution pre-cooled on ice in step (1). Rapid tissue low-temperature crushing homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Add steel balls for crushing. Place the centrifuge tube on the crushing splint and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。将离心管插于冰中，静置孵育8分钟。3) Lyse the cells. Place the centrifuge tube in ice and incubate for 8 minutes.
4)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(3)中的裂解液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。4) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope by using DAPI staining solution: Take 5 μL of the lysate in step (3), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei.
5)RNA质检。使用植物总RNA提取试剂盒(天根生化科技有限公司，DP432)抽提步骤3中细胞核总RNA。使用Agilent 2100生物分析仪质检RNA。结果见表45) RNA quality inspection. The total RNA of the nuclei in step 3 was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP432). RNA quality inspection was performed using an Agilent 2100 bioanalyzer. The results are shown in Table 4
对比实施例28Comparative Example 28
本实施例依照使用破碎仪破碎猕猴桃根组织，KR10裂解液裂解成分为2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(NanjingReagent,8047-15-2)、0.6U/mLRNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATEBIOLOGY,AG11613)。其余操作与对比实施例25相同。结果见表5In this example, the kiwifruit root tissue was broken using a crusher, and the KR10 lysis solution contained 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.6 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613). The remaining operations were the same as those in Comparative Example 25. The results are shown in Table 5
对比实施例29Comparative Example 29
本实施例依照使用70μm(FALCON,352350)筛网过滤两次细胞核悬液。This example uses a 70 μm (FALCON, 352350) mesh to filter the cell nucleus suspension twice.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于70μm滤网(FALCON,352350)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 70 μm filter (FALCON, 352350). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于70μm滤网(FALCON,352350)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 70 μm filter (FALCON, 352350). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(5)中的滤液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表56) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 μL of the filtrate in step (5), mix it with DAPI stain in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 5
对比实施例30Comparative Example 30
本实施例依照使用30μm(Miltenyi Biotec,130-110-915)筛网过滤细胞核悬液1次，其余操作与对比实施例29相同。结果见表5This example uses a 30 μm (Miltenyi Biotec, 130-110-915) mesh to filter the cell nucleus suspension once, and the rest of the operations are the same as those in Comparative Example 29. The results are shown in Table 5
对比实施例31Comparative Example 31
本实施例依照使用30μm(Miltenyi Biotec,130-110-915)筛网过滤细胞核悬液两次，其余操作与对比实施例29相同。结果见表5This example uses a 30 μm (Miltenyi Biotec, 130-110-915) mesh to filter the cell nucleus suspension twice, and the rest of the operations are the same as those in Comparative Example 29. The results are shown in Table 5
对比实施例32Comparative Example 32
本实施例依照使用40μm(FALCON,352340)筛网过滤细胞核悬液，其余操作与对比实施例29相同。结果见表5This example uses a 40 μm (FALCON, 352340) sieve to filter the cell nucleus suspension, and the rest of the operation is the same as that of Comparative Example 29. The results are shown in Table 5
对比实施例33Comparative Example 33
本实施例依照使用40μm(FALCON,352340)筛网过滤细胞核悬液两次，其余操作与对比实施例29相同。结果见表5In this example, the cell nucleus suspension was filtered twice using a 40 μm (FALCON, 352340) sieve, and the rest of the operation was the same as in Comparative Example 29. The results are shown in Table 5
对比实施例34Comparative Example 34
本实施例依照使用70μm(FALCON,352350)筛网过滤去除草酸钙晶体。This example uses a 70 μm (FALCON, 352350) mesh to filter out calcium oxalate crystals.
1)配置裂解液。依照以下成分配置KR裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)过滤。将步骤(5)中的滤液全部使用70μm(FALCON,352350)筛网过滤，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。6) Filtration. Filter all the filtrate in step (5) using a 70 μm (FALCON, 352350) mesh, collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
7)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：取5μL步骤(6)中的滤液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表67) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination with DAPI staining solution: Take 5 μL of the filtrate in step (6), mix it with DAPI staining solution in a ratio of 1:1, use a pipette to draw 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例35Comparative Example 35
本实施例依照使用40μm(FALCON,352340)筛网过滤去除草酸钙晶体，其余操作与对比实施例32相同。结果见表6This example uses a 40 μm (FALCON, 352340) sieve to filter and remove calcium oxalate crystals, and the rest of the operation is the same as that of Comparative Example 32. The results are shown in Table 6
对比实施例36Comparative Example 36
本实施例依照使用30μm(Miltenyi Biotec,130-110-915)筛网过滤去除草酸钙晶体，其余操作与对比实施例32相同。结果见表6This example uses a 30 μm (Miltenyi Biotec, 130-110-915) sieve to filter and remove calcium oxalate crystals, and the rest of the operation is the same as that of Comparative Example 32. The results are shown in Table 6
对比实施例37Comparative Example 37
本实施例依照使用100×g差速离心法去除草酸钙晶体。This example is based on the removal of calcium oxalate crystals using 100×g differential centrifugation.
1)配置裂解液。依照以下成分配置KR裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)差速离心。将步骤(5)中的滤液补充预冷的PBS至10mL，4℃、100×g、10min离心。离心结束后，上清转移至新的离心管中，沉淀使用3mL预冷的PBS重悬。6) Differential centrifugation. Add pre-cooled PBS to the filtrate in step (5) to 10 mL and centrifuge at 4°C, 100×g, for 10 min. After centrifugation, transfer the supernatant to a new centrifuge tube and resuspend the precipitate in 3 mL of pre-cooled PBS.
8)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：分别取5μL步骤(6)中的上清液与重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表68) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by microscopic examination with DAPI stain: Take 5 μL of the supernatant and resuspension in step (6), mix them with DAPI stain in a ratio of 1:1, use a pipette to take 10 μL and add it to the sample well of the hemocytometer for counting, and observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例38Comparative Example 38
本实施例依照使用密度梯度离心法去除草酸钙晶体。This example is based on the use of density gradient centrifugation to remove calcium oxalate crystals.
1)配置裂解液。依照以下成分配置KR裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)密度梯度离心。分别配置50％(m/v)蔗糖溶液、40％(m/v)蔗糖溶液、20％(m/v)蔗糖溶液、15％(m/v)蔗糖溶液。将步骤(5)所得滤液，4℃、500×g、10min离心，离心结束后弃去上清液，使用6mL15％(m/v)蔗糖溶液重悬细胞核沉淀。在Polyallomer离心管(Beckman,355631)内从下到上依次加入50％(m/v)蔗糖溶液、40％(m/v)蔗糖溶液、20％(m/v)蔗糖溶液，之后将6mL细胞核溶液缓缓加在最上层，形成密度梯度。18000rpm离心90min，分别取50％(m/v)蔗糖溶液层、40％(m/v)蔗糖溶液层和20％(m/v)蔗糖溶液层于三个新的离心管中。6) Density gradient centrifugation. Prepare 50% (m/v) sucrose solution, 40% (m/v) sucrose solution, 20% (m/v) sucrose solution, and 15% (m/v) sucrose solution respectively. Centrifuge the filtrate obtained in step (5) at 4°C, 500×g, and 10 min. After centrifugation, discard the supernatant and resuspend the cell nucleus pellet with 6 mL of 15% (m/v) sucrose solution. Add 50% (m/v) sucrose solution, 40% (m/v) sucrose solution, and 20% (m/v) sucrose solution in a Polyallomer centrifuge tube (Beckman, 355631) from bottom to top, and then slowly add 6 mL of cell nucleus solution to the top layer to form a density gradient. Centrifuge at 18000rpm for 90 min, and take the 50% (m/v) sucrose solution layer, the 40% (m/v) sucrose solution layer, and the 20% (m/v) sucrose solution layer into three new centrifuge tubes.
7)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：分别取5μL步骤(6)中的50％(m/v)蔗糖溶液层、40％(m/v)蔗糖溶液层和20％(m/v)蔗糖溶液层，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表67) Microscopic examination and counting. Use DAPI staining solution to calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope: Take 5 μL of the 50% (m/v) sucrose solution layer, 40% (m/v) sucrose solution layer, and 20% (m/v) sucrose solution layer in step (6), respectively, and mix them with DAPI staining solution in a 1:1 ratio. Use a pipette to draw 10ul and add it to the sample well of the blood cell counting plate for counting. Observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例39Comparative Example 39
本实施例依照使用流式细胞术去除草酸钙晶体。This example is based on the removal of calcium oxalate crystals using flow cytometry.
1)配置裂解液。依照以下成分配置KR裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare KR lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)流式细胞仪分选。在步骤(5)所得滤液中加入DAPI染液，DAPI的终浓度为5μg/mL。设置分选条件为紫外激发光下，蓝色荧光信号(+)收集。6) Flow cytometer sorting. Add DAPI dye to the filtrate obtained in step (5), and the final concentration of DAPI is 5 μg/mL. Set the sorting condition to collect blue fluorescence signal (+) under ultraviolet excitation light.
7)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：分别取5μL步骤(6)中收集液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表67) Microscopic examination and counting. Use DAPI staining solution to calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio under a microscope: take 5 μL of the collected solution in step (6) and mix it with DAPI staining solution in a ratio of 1:1. Use a pipette to take 10 ul and add it to the sample well of the blood cell counting plate for counting. Observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例40Comparative Example 40
本实施例依照使用MS分选柱去除草酸钙晶体。This example is based on the use of an MS sorting column to remove calcium oxalate crystals.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：分别取5μL步骤(6)中收集液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表68) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 μL of the collected solution in step (6) and mix it with DAPI stain in a ratio of 1:1. Use a pipette to take 10 μL and add it to the sample well of the blood cell counting plate for counting. Observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例41Comparative Example 41
本实施例依照使用LS分选柱去除草酸钙晶体。This example is based on the use of LS sorting columns to remove calcium oxalate crystals.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入LS Column(Miltenyi,130-042-401)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the LS Column (Miltenyi, 130-042-401), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
9)镜检计数。通过DAPI染液，显微镜下镜检计算细胞核浓度、结团比例、杂质比例：分别取5μL步骤(6)中收集液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表69) Microscopic examination and counting. Calculate the cell nucleus concentration, agglomeration ratio, and impurity ratio by using DAPI stain under a microscope: Take 5 μL of the collected solution in step (6) and mix it with DAPI stain in a ratio of 1:1. Use a pipette to take 10 μL and add it to the sample well of the blood cell counting plate for counting. Observe the number of cell nuclei in the counting area under a microscope. Fluorescence is displayed as cell nuclei. The results are shown in Table 6
对比实施例42Comparative Example 42
本实施例洗涤条件为100×g、4℃、10min洗涤一次。The washing conditions in this embodiment are 100×g, 4° C., and 10 min per wash.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，100×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 100×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
9)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量上清液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表79) Quality inspection, microscopic examination and counting. The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-spectrophotometer and a fluorescence spectrophotometer. The live cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated under a microscope using DAPI stain: 5 μL of the resuspension in step (8) was mixed with DAPI stain in a 1:1 ratio, 10 ul was aspirated with a pipette and added to the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was shown as cell nuclei. The results are shown in Table 7
对比实施例43Comparative Example 43
本实施例洗涤条件为1000×g、4℃、10min洗涤一次。The washing conditions in this embodiment are 1000×g, 4° C., and 10 min per wash.
1)配置裂解液。依照以下成分配置裂解液： 2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，1000×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 1000×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
9)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量上清液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。]结果见表7。9) Quality inspection, microscopic examination and counting. The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-spectrophotometer and a fluorescence spectrophotometer. The live cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated by microscopic examination using DAPI staining solution: 5 μL of the resuspension in step (8) was mixed with DAPI staining solution in a 1:1 ratio, 10 ul was aspirated with a pipette and added to the sample well of the hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was displayed as cell nuclei.] The results are shown in Table 7.
对比实施例44Comparative Example 44
本实施例洗涤条件为300×g、4℃、10min洗涤一次。The washing conditions in this embodiment are 300×g, 4° C., and 10 min per wash.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
9)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量上清液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(8)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表79) Quality inspection, microscopic examination and counting. The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-spectrophotometer and a fluorescence spectrophotometer. The live cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated under a microscope using DAPI stain: 5 μL of the resuspension in step (8) was mixed with DAPI stain in a 1:1 ratio, 10 ul was aspirated with a pipette and added to the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was shown as cell nuclei. The results are shown in Table 7
对比实施例45Comparative Example 45
本实施例洗涤条件为300×g、4℃、10min洗涤三次。The washing conditions in this example are 300×g, 4° C., and 10 min for three washes.
1)配置裂解液。依照以下成分配置裂解液：2％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15mM柠檬酸钠、0.2％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。将配置好的裂解液置于冰上进行预冷。1) Prepare lysis buffer. Prepare lysis buffer according to the following ingredients: 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (mouse), ACCURATE BIOLOGY, AG11613). Pre-cool the prepared lysis buffer on ice.
2)破碎样本。将50mg猕猴桃根组织样本转入2mL离心管(Axygen,MCT-200-C)中。加入步骤(1)中冰上预冷的裂解液1mL，加入打碎用的钢珠。将离心管置于打碎用夹板并拧紧扳手，调节仪器转速为1200rpm，180秒。启动仪器进行组织破碎。2) Crush the sample. Transfer 50 mg of kiwifruit root tissue sample into a 2 mL centrifuge tube (Axygen, MCT-200-C). Add 1 mL of the lysis solution pre-cooled on ice in step (1) and steel beads for crushing. Place the centrifuge tube on the crushing clamp and tighten the wrench, adjust the instrument speed to 1200 rpm for 180 seconds. Start the instrument to crush the tissue.
3)裂解细胞。待仪器完全停止后，将离心管取出后插于冰中，静置孵育7分钟。3) Lyse cells. After the instrument stops completely, take out the centrifuge tube and put it in ice for 7 minutes.
4)过滤。用移液器将步骤(3)中的组织裂解液转移到一个新的15mL离心管(Corning,430790)中。向离心管中加入9mL冰上预冷的PBS(Gibco,10010-031)。于冰上静置1分钟。使用移液器将裂解液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。过滤液收集到一新的50mL离心管(Corning,430828)中。注意操作均应在冰上进行。4) Filtration. Use a pipette to transfer the tissue lysate in step (3) to a new 15 mL centrifuge tube (Corning, 430790). Add 9 mL of ice-cold PBS (Gibco, 10010-031) to the centrifuge tube. Let stand on ice for 1 minute. Use a pipette to aspirate the lysate gradually and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 50 mL centrifuge tube (Corning, 430828). Note that all operations should be performed on ice.
5)过滤。使用移液器，将步骤(4)中的过滤液逐次吸出，于40μm滤网(FALCON,352340)进行过滤。将过滤液收集到一新的15mL离心管中。注意操作均应在冰上进行。5) Filtration. Use a pipette to gradually aspirate the filtrate from step (4) and filter it through a 40 μm filter (FALCON, 352340). Collect the filtrate into a new 15 mL centrifuge tube. Note that all operations should be performed on ice.
6)离心收集沉淀。将步骤(5)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用3mL冰上预冷的PBS进行重悬。6) Collect the precipitate by centrifugation. Place the centrifuge tube in step (5) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 3 mL of ice-cold PBS.
7)过滤。将步骤(6)中的3mL重悬液全部加入MS Column(Miltenyi,130-042-201)中，将过滤液收集到一新的15mL离心管中，于冰上静置过滤，待液体完全过滤到15mL离心管中为止。7) Filtration. Add all 3 mL of the resuspension in step (6) to the MS Column (Miltenyi, 130-042-201), collect the filtrate into a new 15 mL centrifuge tube, and filter on ice until the liquid is completely filtered into the 15 mL centrifuge tube.
8)离心收集沉淀。将步骤(7)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬。8) Collect the precipitate by centrifugation. Place the centrifuge tube in step (7) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 10 mL of ice-cold PBS.
9)离心收集沉淀。将步骤(8)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用10mL冰上预冷的PBS进行重悬。9) Collect the precipitate by centrifugation. Place the centrifuge tube in step (8) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant carefully, and resuspend the precipitate in 10 mL of ice-cold PBS.
10)离心收集沉淀。将步骤(9)中的离心管置于离心机中，4℃下，300×g离心10分钟。离心完成后，小心取出离心管，将上清小心的倒出，沉淀使用100μL冰上预冷的PBS进行重悬。10) Collect the precipitate by centrifugation. Place the centrifuge tube in step (9) in a centrifuge and centrifuge at 300×g for 10 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube, pour out the supernatant, and resuspend the precipitate in 100 μL of ice-cold PBS.
11)质检、镜检计数。吸取上清液使用NanoDrop微量分光光度计和荧光分光光度计测量上清液中RNA的浓度。通过DAPI染液，显微镜下镜检计算活细胞核浓度、结团比例、杂质比例：取5μL步骤(10)中的重悬液，与DAPI染液1:1进行混匀，用移液器吸取10ul加到血球计数板的加样孔中计数，显微镜观察计数区的细胞核个数。荧光显示为细胞核。结果见表711) Quality inspection, microscopic examination and counting. The supernatant was aspirated and the RNA concentration in the supernatant was measured using a NanoDrop micro-spectrophotometer and a fluorescence spectrophotometer. The live cell nucleus concentration, agglomeration ratio, and impurity ratio were calculated under a microscope using DAPI stain: 5 μL of the resuspension in step (10) was mixed with DAPI stain in a 1:1 ratio, 10 ul was aspirated with a pipette and added to the sample well of a hemocytometer for counting, and the number of cell nuclei in the counting area was observed under a microscope. Fluorescence was shown as cell nuclei. The results are shown in Table 7
对比实施例46Comparative Example 46
本实施例实验样本为50mg拟南芥叶片，具体操作与实施例1相同。结果见表8The experimental sample of this example is 50 mg of Arabidopsis leaves, and the specific operation is the same as that of Example 1. The results are shown in Table 8
对比实施例47Comparative Example 47
本实施例实验样本为50mg拟南芥根，具体操作与实施例1相同。结果见表8The experimental sample of this example is 50 mg of Arabidopsis roots, and the specific operation is the same as that of Example 1. The results are shown in Table 8
对比实施例48Comparative Example 48
本实施例实验样本为50mg玉米胚，具体操作与实施例1相同。结果见表8The experimental sample of this example is 50 mg corn embryo, and the specific operation is the same as that of Example 1. The results are shown in Table 8
表1实施例1～4结果Table 1 Results of Examples 1 to 4
本发明描述了一种适用于猕猴桃根组织单细胞核测序的方法，包括猕猴桃根组织细胞核提取的方法以及细胞核悬液优化方法。本发明所提供的技术方法可以将单细胞测序技术引入猕猴桃分子机制及育种的相关研究中，可以有效且高效的促进研究的进程。The present invention describes a method suitable for sequencing single cell nuclei of kiwifruit root tissue, including a method for extracting cell nuclei from kiwifruit root tissue and a method for optimizing cell nuclei suspension. The technical method provided by the present invention can introduce single cell sequencing technology into the relevant research on kiwifruit molecular mechanism and breeding, and can effectively and efficiently promote the progress of research.
本发明设计的一种适用于猕猴桃根组织的单细胞核制备方法主要包括一种裂解液，其成分为2％～3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15～25mM柠檬酸钠、0.2～0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2～0.4U/mL RNaseinhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)。通过破碎样本、裂解细胞从组织中提取细胞核，再经筛网过滤、离心洗涤等悬液优化处理后得到数量大于1×106个、碎片率小于10％、结团率小于4％的细胞核悬液，满足10×单细胞测序对于细胞核悬液的质量要求。达到了使用高通量单细胞测序技术进行猕猴桃根组织研究的目的。The single cell nucleus preparation method for kiwifruit root tissue designed by the present invention mainly includes a lysate, whose components are 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613). The cell nucleus is extracted from the tissue by crushing the sample and lysing the cells, and then the cell nucleus suspension with a number greater than 1×106, a fragmentation rate less than 10%, and a clumping rate less than 4% is obtained after the suspension is optimized by sieve filtration, centrifugal washing, etc., which meets the quality requirements of 10× single cell sequencing for the cell nucleus suspension. The purpose of using high-throughput single cell sequencing technology to study kiwifruit root tissue is achieved.
为了得到本发明中的一种适用于猕猴桃根组织单细胞核测序的方法，进行了从裂解液配方、实验操作步骤、优化方法等方面的一系列探索，通过查阅资料与验证最终得到了本发明中的内容。首先通过对比实施例1～3，使用《Identification ofOpen ChromatinRegions in Plant Genomes UsingATAC-Seq》中的方法分别进行了拟南芥叶片、猕猴桃叶片、猕猴桃根组织细胞核悬液制备，结果显示该文献中的方法仅可以完成拟南芥叶片组织细胞核悬液的制备，无法完成猕猴桃叶片与猕猴桃根组织的细胞核悬液制备；且制备得拟南芥叶片细胞核悬液中杂质占比稍高，虽满足单细胞测序对样本的最低要求，但进行单细胞测序存在风险。在对比实施例4～6中，使用《Isolation of Plant Root Nuclei forSingle Cell RNA Sequencing》的方法使用现有商业化试剂盒提取细胞核实验进行了拟南芥根组织、猕猴桃叶片组织、猕猴桃根组织的细胞核悬液制备实验，结果显示，文献中所属方法仅可以完成拟南芥根组织细胞核悬液制备，无法完成猕猴桃叶片组织细胞核悬液制备，结果显示现有的商业化试剂盒提取得到的猕猴桃根细胞核数量极少，无法成功制备满足单细胞测序的猕猴桃根组织细胞核悬液。上述的两种方法均表现出，制备出细胞核数量少，杂质极多的现象。裂解液充分裂解组织的同时有合适的缓冲液保障细胞核完整性是保障提取细胞核数量关键。In order to obtain a method suitable for single-cell nuclear sequencing of kiwifruit root tissue in the present invention, a series of explorations were conducted from the aspects of lysate formula, experimental operation steps, optimization methods, etc., and the content of the present invention was finally obtained by consulting materials and verification. First, by comparing Examples 1 to 3, the method in "Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq" was used to prepare the nuclear suspension of Arabidopsis leaves, kiwifruit leaves, and kiwifruit root tissues, respectively. The results showed that the method in the document can only complete the preparation of the nuclear suspension of Arabidopsis leaf tissue, and cannot complete the preparation of the nuclear suspension of kiwifruit leaves and kiwifruit root tissues; and the proportion of impurities in the prepared Arabidopsis leaf nuclear suspension is slightly higher. Although it meets the minimum requirements for single-cell sequencing for samples, there are risks in single-cell sequencing. In comparative examples 4 to 6, the method of "Isolation of Plant Root Nuclei for Single Cell RNA Sequencing" was used to extract nuclei using existing commercial kits to prepare nucleus suspensions of Arabidopsis root tissue, kiwi leaf tissue, and kiwi root tissue. The results showed that the method in the literature can only complete the preparation of nucleus suspensions of Arabidopsis root tissue, but cannot complete the preparation of nucleus suspensions of kiwi leaf tissue. The results showed that the number of kiwi root nuclei extracted by the existing commercial kits was very small, and it was impossible to successfully prepare a kiwi root tissue nucleus suspension that met the requirements of single cell sequencing. Both of the above methods showed the phenomenon of preparing a small number of nuclei and a large number of impurities. The key to ensuring the number of extracted nuclei is that the lysis solution fully lyses the tissue while having a suitable buffer to ensure the integrity of the nuclei.
首先通过对比实施例7～20，对适合猕猴桃根组织的缓冲体系进行了探索，结果如表1所示，现有方案中的缓冲体系无法适用于猕猴桃根组织细胞核提取实验；猕猴桃生长于野外，细胞生长机制与模式生物大不相同，猕猴桃根细胞中会富集草酸钙、醛类等。相比与模式生物拟南芥等需要更强缓冲能力的缓冲组分，在常用的缓冲组分中，Tris-HCl、MOPS等均不适用。提高浓度会促进Tris与溶液中醛以及各种酶的反应，导致裂解体系不稳定。最终经过探索选择了L-酒石酸与柠檬酸钠一起构建的缓冲体系。L-酒石酸常用于饮料和其他食品的酸味剂，有强的缓冲能力，在本发明中起主要缓冲作用；同时酒石酸还有较强的还原性，在本发明中发挥着对细胞核内核酸的保护作用。对比实施例结果显示缓冲组分浓度低时，无法保持缓冲液pH稳定，细胞核由于缓冲液pH变化而破碎；缓冲组分浓度高时，细胞核因为缓冲液中离子浓度高而发生结团，导致实验失败。因此本发明中细胞核制备缓冲组分为2％～3％(v/v)L-酒石酸(Nanjing Reagent,C0681014023)、15～25mM柠檬酸钠。First, by comparing Examples 7 to 20, the buffer system suitable for kiwifruit root tissue was explored. The results are shown in Table 1. The buffer system in the existing scheme is not suitable for the kiwifruit root tissue cell nucleus extraction experiment; kiwifruit grows in the wild, and the cell growth mechanism is very different from that of model organisms. Calcium oxalate, aldehydes, etc. are enriched in kiwifruit root cells. Compared with the buffer components that require stronger buffering capacity such as model organisms Arabidopsis, among the commonly used buffer components, Tris-HCl, MOPS, etc. are not applicable. Increasing the concentration will promote the reaction of Tris with aldehydes and various enzymes in the solution, resulting in an unstable lysis system. Finally, after exploration, a buffer system constructed with L-tartaric acid and sodium citrate was selected. L-tartaric acid is commonly used as an acidulant for beverages and other foods, has a strong buffering capacity, and plays a major buffering role in the present invention; at the same time, tartaric acid also has a strong reducing property, and plays a protective role for nucleic acids in the cell nucleus in the present invention. The results of the comparative example show that when the concentration of the buffer component is low, the pH of the buffer cannot be kept stable, and the nucleus is broken due to the change of the pH of the buffer; when the concentration of the buffer component is high, the nucleus agglomerates due to the high ion concentration in the buffer, resulting in experimental failure. Therefore, the buffer component for preparing the nucleus in the present invention is 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15 to 25 mM sodium citrate.
提取植物细胞核需要破坏细胞膜，细胞膜的基本结构是磷脂双分子层，常用的是使用去污剂破坏磷脂双分子层可以达到释放细胞核的目的。在对比实施例7～10中进行了裂解液配方的探索，通过不同性质不同工作原理的裂解组分进行实验，结果显示SDS(十二烷基硫酸钠)、脱氧胆酸钠强去污剂作为裂解成分时，大量细胞核破碎，无法收集到足量完整的细胞核；洋地黄皂苷弱去污剂作为裂解成分时，由于洋地黄皂苷无法溶解膜蛋白，细胞膜结构无法得到充分破坏，可以收集到相对多一些的细胞核但仍无法满足单细胞测序对细胞核数量的要求。Triton X-100属于温和的去污剂来源于聚氧乙烯，并含有一个烷苯基疏水基团，结合脂分子的能力介于十二烷基硫酸钠和洋地黄皂苷之间，且可以作为膜蛋白的助溶剂，裂解得到的细胞核数量较于洋地黄皂更多，但Triton X-100作用于细胞膜的同时还会对核膜造成损伤，仍无法得到足量的细胞核进行单细胞核测序实验。常用的去污剂溶解细胞膜制备细胞核的方法不适用于猕猴桃根组织的细胞核制备实验，皂素常用于医药原料，主要用于激素类药物的合成。皂素是由皂苷和糖、糖醛酸或其他有机酸所组成，皂苷赋予了皂素乳化脂分子的功能。对比实施例结果显示，皂素作为裂解组分时，细胞核产量相比于SDS、Triton X-100等有极其显著的提升；同时因为皂素不会导致蛋白变性，所以皂素作为裂解组分是对细胞核核膜不会有损伤。对比实施例结果显示，皂素浓度低于2％时，细胞核数量会明显下降，因为皂素本身含有糖于有机酸等，皂素浓度高于4％时，会影响裂解体系的稳定性，细胞核数量减少。因此本发明裂解液中皂素的浓度范围是2％～4％(m/v)。Extracting plant cell nuclei requires destroying the cell membrane, and the basic structure of the cell membrane is a phospholipid bilayer. It is common to use detergents to destroy the phospholipid bilayer to achieve the purpose of releasing the cell nucleus. In comparative examples 7 to 10, the lysate formula was explored, and experiments were conducted using lysate components with different properties and working principles. The results showed that when strong detergents SDS (sodium dodecyl sulfate) and sodium deoxycholate were used as lysate components, a large number of cell nuclei were broken, and it was impossible to collect a sufficient number of intact cell nuclei; when weak detergent digitonin was used as a lysate component, since digitonin could not dissolve membrane proteins, the cell membrane structure could not be fully destroyed, and relatively more cell nuclei could be collected, but the requirement for the number of cell nuclei for single-cell sequencing could still not be met. Triton X-100 is a mild detergent derived from polyethylene oxide and contains an alkylphenyl hydrophobic group. Its ability to bind lipid molecules is between sodium dodecyl sulfate and digitonin, and it can be used as a cosolvent for membrane proteins. The number of cell nuclei obtained by lysis is more than that of digitonin, but Triton X-100 will damage the nuclear membrane while acting on the cell membrane, and it is still impossible to obtain enough cell nuclei for single cell nuclear sequencing experiments. The commonly used method of dissolving cell membranes with detergents to prepare cell nuclei is not suitable for the cell nucleus preparation experiment of kiwi root tissue. Saponin is commonly used in pharmaceutical raw materials, mainly for the synthesis of hormone drugs. Saponin is composed of saponin and sugar, uronic acid or other organic acids, and saponin gives saponin the function of emulsifying lipid molecules. The results of the comparative example show that when saponin is used as a lysis component, the cell nucleus yield is significantly improved compared with SDS, Triton X-100, etc.; at the same time, because saponin does not cause protein denaturation, saponin as a lysis component will not damage the cell nucleus and nuclear membrane. The results of the comparative example show that when the saponin concentration is lower than 2%, the number of cell nuclei will decrease significantly, because saponin itself contains sugars and organic acids, etc. When the saponin concentration is higher than 4%, it will affect the stability of the lysis system and reduce the number of cell nuclei. Therefore, the concentration range of saponin in the lysis solution of the present invention is 2% to 4% (m/v).
表2对比实施例1～20Table 2 Comparative Examples 1 to 20
植物细胞膜外还有细胞壁包裹，细胞壁是由纤维素、半纤维素、果胶等成分构成，猕猴桃植株为大型落叶藤本，细胞壁的支撑强度要远远高于拟南芥，因此在破碎细胞壁的方法上，需要更强的破碎条件来帮助得到更多的细胞核。接下来通过对比实施例21～25，通过比较不同的处理方法包括刀片切碎、剪刀剪碎、匀浆化、破碎仪破碎、研钵研磨等对细胞核释放的影响，结果显示，刀片切碎、剪刀剪碎、研钵研磨的实验方法得到的细胞核数量都低于匀浆方法，研钵液氮研磨是组织破碎最充分的办法，但是研钵研磨破环细胞壁的同时对细胞核也造成了破坏。分析对比实施例结果得出，破碎仪破碎的方式辅助裂解液充分释放细胞核的同时对细胞核破坏更少，是提取猕猴桃根组织细胞核的最佳方法。Plant cell membranes are wrapped by cell walls, which are composed of cellulose, hemicellulose, pectin and other components. Kiwi plants are large deciduous vines, and the supporting strength of the cell walls is much higher than that of Arabidopsis. Therefore, in the method of breaking the cell walls, stronger breaking conditions are needed to help obtain more nuclei. Next, by comparing the effects of different treatment methods including blade chopping, scissors chopping, homogenization, crusher crushing, mortar grinding, etc. on the release of nuclei, the results show that the number of nuclei obtained by the experimental methods of blade chopping, scissors chopping, and mortar grinding is lower than that of the homogenization method. Mortar liquid nitrogen grinding is the most sufficient way to break tissue, but mortar grinding also damages the nucleus while breaking the cell wall. The results of the comparative examples show that the crusher crushing method assists the lysate to fully release the nucleus while causing less damage to the nucleus, which is the best method for extracting the nucleus of kiwi root tissue.
表3对比实施例21～25Table 3 Comparative Examples 21 to 25
植物细胞核进行单细胞转录组实验时，需要保障细胞内mRNA完整，而细胞破碎后，胞质中的RNA酶释放到缓冲体系中会降解mRNA，因此需要RNA酶抑制剂和还原剂保护细胞核mRNA，不同物种、组织中的RNA酶含量差异较大，对比实施例26～28结果显示，针对50～100mg猕猴桃根组织，0.2～0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATE BIOLOGY,AG11613)可以很好的保护mRNA完整性，提高RNA酶抑制剂得到的结果无明显差异，还会造成成本提高和试剂的浪费，因此KR裂解液中RNA酶抑制剂的浓度为0.2～0.4U/mL。When single-cell transcriptome experiments are performed on plant cell nuclei, the integrity of intracellular mRNA needs to be ensured. After the cells are broken, the RNase in the cytoplasm is released into the buffer system to degrade the mRNA. Therefore, RNase inhibitors and reducing agents are needed to protect nuclear mRNA. The RNase content in different species and tissues varies greatly. The results of comparative examples 26 to 28 show that for 50 to 100 mg of kiwi root tissue, 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG11613) can well protect the integrity of mRNA. There is no significant difference in the results obtained by increasing the RNase inhibitor, which will also increase costs and waste reagents. Therefore, the concentration of RNase inhibitor in KR lysate is 0.2 to 0.4 U/mL.
表4.对比实施例26～28Table 4. Comparative Examples 26 to 28
组织经过裂解液处理后提取得到的粗细胞核悬液含有大量的细胞碎片等杂质和细胞破碎释放的mRNA、细胞器等。这种条件下的细胞核悬液存在大的碎片无法进行单细胞测序实验，需要去除大的碎片后才能满足单细胞测序的要求，首先过滤去除较大的组织和细胞碎片，通过对比实施例29～33，验证了不同孔径筛网、不同过滤次数，过滤粗细胞核悬液的得率与除杂效率，对比实施例29结果显示70μm筛网不能很好的达到除杂的目的，经70μm筛网过滤后的悬液杂质减少不明显，两次过滤仍然无明显效果。破碎仪破碎的样本大于70μm的碎片较少，在对比实施例30～31中，验证了30μm孔径筛网过滤猕猴桃根粗细胞核悬液的效果，结果显示，30μm的细胞筛会导致细胞核的大量损失，且过滤过程受碎片率高的影响，过滤所需时间长，加大了细胞核内RNA降解的风险，因此30μm筛网不能作为猕猴桃根粗细胞核悬液纯化的方法。通过对比实施例32～33结果可知，40μm细胞筛网可以快速的去除粗细胞核悬液中的杂质，纯化猕猴桃根粗细胞核悬液最好的方法是40μm细胞筛网过滤两次，除杂效率高达80％，同时细胞核得率大于70％，这个选择既快速有效降低了碎片率，又有着更高的细胞核得率。The crude cell nucleus suspension extracted from the tissue after being treated with a lysate contains a large amount of impurities such as cell fragments and mRNA and organelles released by cell fragmentation. Under such conditions, the cell nucleus suspension has large fragments and cannot be used for single-cell sequencing experiments. The large fragments need to be removed to meet the requirements of single-cell sequencing. First, the larger tissue and cell fragments are filtered to remove them. By comparing Examples 29 to 33, the yield and impurity removal efficiency of the crude cell nucleus suspension filtered with different aperture meshes and different filtering times are verified. The results of Comparative Example 29 show that the 70μm mesh cannot achieve the purpose of impurity removal very well. The impurities of the suspension after filtration with the 70μm mesh are not significantly reduced, and there is still no obvious effect after two filtrations. The sample broken by the crusher has fewer fragments larger than 70 μm. In comparative examples 30-31, the effect of filtering the crude cell nucleus suspension of kiwifruit root with a 30 μm aperture sieve was verified. The results showed that the 30 μm cell sieve would lead to a large loss of cell nuclei, and the filtration process was affected by the high fragmentation rate, and the filtration time was long, which increased the risk of RNA degradation in the cell nucleus. Therefore, the 30 μm sieve could not be used as a method for purifying the crude cell nucleus suspension of kiwifruit root. The results of comparative examples 32-33 show that the 40 μm cell sieve can quickly remove impurities in the crude cell nucleus suspension. The best method for purifying the crude cell nucleus suspension of kiwifruit root is to filter twice with a 40 μm cell sieve, with an impurity removal efficiency of up to 80%, and a cell nucleus yield of more than 70%. This choice not only quickly and effectively reduces the fragmentation rate, but also has a higher cell nucleus yield.
表5对比实施例29～33Table 5 Comparative Examples 29 to 33
[[0719]猕猴桃植株在生长过程中，会在细胞/组织内富集大量的草酸钙针状晶体，这些草酸钙针状晶体是构成植物防御和自我保护的重要组成部分。而这些草酸钙晶体在组织破碎后会释放到细胞核悬液中(图3所示为细胞核悬液中草酸钙针状晶体)，筛网过滤后的细胞悬液中还存在针状结晶体，在筛网过滤中，无论是30μm还是70μm都无法有效的完成对该类杂质的过滤。而草酸钙晶体的存在会导致无法开展单细胞测序，草酸钙晶体在猕猴桃根组织中以针状晶体存在，粗细长短不一，根据显微镜观察可知草酸钙晶体长度大于70μm。通过对比实施例34验证70μm细胞筛网去除草酸钙晶体的效果，结果显示70μm细胞筛无法有效去除草酸钙针状晶体。通过对比实施例35～36验证了40μm、30μm筛网去除草酸钙晶体的效果，结果显示，减小筛网孔径仍然不能去除草酸钙晶体。草酸钙针状晶体很容易伴随液流穿过筛网，因此筛网过滤的方法无法去除草酸钙针状晶体。接下来通过对比实施例37～39，分别验证了差速离心法、密度梯度离心法、流式细胞术方法去除草酸钙晶体的效果，结果显示，差速离心法无法达到去除草酸钙晶体的作用，且会导致细胞核大量损失；密度梯度离心会导致细胞核的大量损失，继续进行单细胞核测序存在风险，杂质的比重与细胞核相近导致了密度梯度离心不能很好的纯化细胞核，差速离心法和密度梯度离心法都不能达到纯化的细胞核的目的。而草酸钙针状晶体的存在会直接导致流式细胞仪堵塞，无法收集足量的细胞核进行实验。对比实施例40～41使用MACS分选柱进行细胞核悬液优化的探索，分选柱(MACS)通常用于磁场下，通过带有磁珠的抗体结合目的细胞膜表面的特异性抗原，从而用于动物特定细胞类型的分选，在本发明中，在非磁场下，利用草酸钙晶体的针状形态特征和分选柱中纳米材料的堆叠的特点，体积小的细胞核可以伴随液流穿过分选柱，针状草酸钙晶体则会被留在分选柱中，无法伴随液流穿过，达到纯化细胞核的目的可以达到除杂目的的同时保障细胞核得率，解决了单细胞核悬液优化的问题。结果显示，通过MS分选柱过滤单细胞核悬液得到的滤液各项指标都满足10×Genmics平台对于单细胞测序的要求。[[0719] During the growth process of kiwifruit plants, a large amount of calcium oxalate needle crystals will be enriched in cells/tissues. These calcium oxalate needle crystals are an important component of plant defense and self-protection. These calcium oxalate crystals will be released into the cell nuclear suspension after tissue fragmentation (Figure 3 shows calcium oxalate needle crystals in the cell nuclear suspension). There are still needle crystals in the cell suspension after screen filtration. In the screen filtration, neither 30μm nor 70μm can effectively complete the filtration of this type of impurities. The presence of calcium oxalate crystals will make it impossible to carry out single-cell sequencing. Calcium oxalate crystals exist in kiwifruit root tissues as needle crystals, with different thicknesses and lengths. According to microscopic observation, it can be seen that the length of calcium oxalate crystals is greater than 70μm. The effect of 70μm cell sieve on removing calcium oxalate crystals was verified by comparative example 34. The results show that 70μm cell sieve cannot effectively remove calcium oxalate needle crystals. The effect of removing calcium oxalate crystals by 40μm and 30μm sieves was verified by comparative examples 35 to 36. The results show that reducing the sieve aperture still cannot remove calcium oxalate crystals. Calcium oxalate needle-shaped crystals can easily pass through the sieve with liquid flow, so the method of sieve filtration cannot remove calcium oxalate needle-shaped crystals. Next, by comparative examples 37 to 39, the effects of removing calcium oxalate crystals by differential centrifugation, density gradient centrifugation, and flow cytometry were verified respectively. The results show that differential centrifugation cannot achieve the effect of removing calcium oxalate crystals, and can cause a large loss of cell nuclei; density gradient centrifugation can cause a large loss of cell nuclei, and there is a risk of continuing single cell nucleus sequencing. The specific gravity of impurities is similar to that of cell nuclei, resulting in that density gradient centrifugation cannot purify cell nuclei well, and both differential centrifugation and density gradient centrifugation cannot achieve the purpose of purifying cell nuclei. The presence of calcium oxalate needle-shaped crystals will directly cause the flow cytometer to be blocked, and it is impossible to collect enough cell nuclei for experiments. Comparative Examples 40 to 41 use MACS sorting columns to explore the optimization of cell nucleus suspensions. Sorting columns (MACS) are usually used in magnetic fields to bind specific antigens on the surface of target cell membranes through antibodies with magnetic beads, so as to be used for sorting specific cell types of animals. In the present invention, in a non-magnetic field, the needle-shaped morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting column are utilized. Small cell nuclei can pass through the sorting column with the liquid flow, while needle-shaped calcium oxalate crystals will be left in the sorting column and cannot pass through with the liquid flow. The purpose of purifying cell nuclei can achieve the purpose of removing impurities while ensuring the cell nucleus yield, solving the problem of optimizing single cell nucleus suspensions. The results show that all indicators of the filtrate obtained by filtering the single cell nucleus suspension through the MS sorting column meet the requirements of the 10×Genmics platform for single-cell sequencing.
表6.对比实施例34～41Table 6. Comparative Examples 34 to 41
完成草酸钙晶体去除后，需要通过洗涤去除细胞核悬液中游离的mRNA提高数据质量，同时需要对细胞核悬液的浓度进行调整，对比实施例42～45中，进行了对洗涤条件的验证。结果显示，过小的离心力会导致细胞核损失较大，过大的离心力会导致细胞核损伤，过多的洗涤次数会导致细胞核破碎；洗涤条件为4℃下，300×g～500×g离心10分钟，洗涤2次，细胞核悬液中游离的mRNA基本去除干净，同时细胞核得率高于其它方法，是适合猕猴桃根组织细胞核悬液的洗涤条件。After the calcium oxalate crystals are removed, the free mRNA in the cell nucleus suspension needs to be removed by washing to improve the data quality. At the same time, the concentration of the cell nucleus suspension needs to be adjusted. In comparative examples 42 to 45, the washing conditions were verified. The results show that too small a centrifugal force will lead to a large loss of cell nuclei, too large a centrifugal force will lead to cell nucleus damage, and too many washing times will lead to cell nucleus fragmentation; the washing conditions are 4°C, 300×g to 500×g centrifugation for 10 minutes, washing twice, and the free mRNA in the cell nucleus suspension is basically removed. At the same time, the cell nucleus yield is higher than other methods, which is suitable for the washing conditions of kiwi root tissue cell nucleus suspension.
表7.对比实施例42～45Table 7. Comparative Examples 42 to 45
不同物种、不同组织的植物细胞壁构成不同，细胞内容物的构成也不同，对比实施例46～48验证了KR裂解液与本专利中的细胞核提取、优化方法制备拟南芥叶片、拟南芥根尖、玉米胚细胞核悬液的结果。结果显示，本专利中的细胞核制备方法，及相应的裂解液配方和细胞核优化方法与猕猴桃根组织一一对应，需要按照步骤先后进行才能完成猕猴桃根组织的细胞核悬液制备，对其它物种和组织不适用。Plant cell walls of different species and tissues have different compositions, and the composition of cell contents is also different. Comparative Examples 46 to 48 verified the results of preparing the nuclear suspension of Arabidopsis leaves, Arabidopsis root tips, and corn embryos using the KR lysate and the cell nucleus extraction and optimization methods in this patent. The results show that the cell nucleus preparation method in this patent, and the corresponding lysate formula and cell nucleus optimization method correspond one-to-one with kiwifruit root tissue, and the steps need to be performed in sequence to complete the preparation of the nuclear suspension of kiwifruit root tissue, which is not applicable to other species and tissues.
表8.对比实施例46～48Table 8. Comparative Examples 46 to 48
综上所述，本发明描述的一种适用于单细胞测序的猕猴桃根组织细胞核制备方法，通过对比实施例1～28得出了KR裂解液其成分为2％～3％(v/v)L-酒石酸(NanjingReagent,C0681014023)、15～25mM柠檬酸钠、0.2～0.4％(m/v)皂素(Nanjing Reagent,8047-15-2)、0.2～0.4U/mL RNase inhibitor(重组型RNase抑制剂(鼠源)，ACCURATEBIOLO；辅助猕猴桃根细胞核释放的方式为破碎仪破碎。通过对比实施例29～41得出了优化细胞核悬液的实验方法：40μm筛网过滤两遍去除大的碎片，MS分选柱去除草酸钙针状晶体；通过对比实施例42～45验证了一种细胞核高得率，去除环境RNA的猕猴桃根组织细胞核悬液洗涤方法，为300～500×g、4℃、10min洗涤两次。对比实施例46～48结果显示本发明所述的KR裂解液、细胞核悬液过滤操作、细胞核悬液洗涤操作仅适用于猕猴桃根组织细胞核悬液制备。In summary, the present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing. By comparing Examples 1 to 28, a KR lysate solution is obtained, which comprises 2% to 3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15 to 25 mM sodium citrate, 0.2 to 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2 to 0.4 U/mL RNase inhibitor (recombinant RNase inhibitor (mouse source), ACCURATEBIOLO; the method of assisting the release of kiwifruit root nuclei is crushing with a crusher. By comparing Examples 29 to 41, an experimental method for optimizing the nucleus suspension was obtained: filtering twice with a 40μm mesh to remove large fragments, and removing calcium oxalate needle crystals with an MS sorting column; by comparing Examples 42 to 45, a method for washing a kiwifruit root tissue nucleus suspension with a high nucleus yield and removal of environmental RNA was verified, which was washed twice at 300 to 500×g, 4°C, and 10min. The results of Comparative Examples 46 to 48 show that the KR lysate, nucleus suspension filtration operation, and nucleus suspension washing operation described in the present invention are only applicable to the preparation of kiwifruit root tissue nucleus suspension.
本发明描述的一种适用于单细胞测序的猕猴桃根组织细胞核制备方法可有效制备开展10x Genomics平台单细胞转录组测序实验所需的细胞核悬液。本发明所述的一种适用于单细胞转录组测序的猕猴桃细胞核悬液制备方法中猕猴桃根、KR裂解液、细胞核悬液过滤操作、细胞核悬液洗涤操作、单细胞转录组测序结果之间是一一对应关系的，即猕猴桃根、KR裂解液、细胞核悬液过滤操作、细胞核悬液洗涤操作、单细胞转录组测序结果存在配伍关系。如，本发明实施例1～4提到的猕猴桃根、KR裂解液、细胞核悬液过滤操作，应当按照本发明中所指出的方法使用，才能有效的制备细胞核悬液，且制备得到的细胞核悬液各项指标能够达到单细胞转录组测序要求。本发明所述的一种适用于单细胞转录组测序的猕猴桃根细胞核悬液制备方法操作科学严谨，重复性高，能够有效地、高效地从猕猴桃根组织中分离得到数量足够多、杂质率低、极低环境RNA的细胞核悬液，且得到的细胞核悬液能够达到10x Genomics平台单细胞转录组测序各项指标。The present invention describes a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, which can effectively prepare the nucleus suspension required for the single-cell transcriptome sequencing experiment of the 10x Genomics platform. In the method for preparing a kiwifruit nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention, there is a one-to-one correspondence between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results, that is, there is a compatibility relationship between the kiwifruit root, KR lysate, nucleus suspension filtration operation, nucleus suspension washing operation, and single-cell transcriptome sequencing results. For example, the kiwifruit root, KR lysate, and nucleus suspension filtration operation mentioned in Examples 1 to 4 of the present invention should be used according to the method specified in the present invention to effectively prepare the nucleus suspension, and the various indicators of the prepared nucleus suspension can meet the requirements of single-cell transcriptome sequencing. The method for preparing a kiwifruit root cell nucleus suspension suitable for single-cell transcriptome sequencing described in the present invention is scientifically rigorous in operation, highly repeatable, and can effectively and efficiently separate a cell nucleus suspension with a sufficient number, low impurity rate, and extremely low environmental RNA from kiwifruit root tissue, and the obtained cell nucleus suspension can meet various indicators of single-cell transcriptome sequencing on the 10x Genomics platform.
本发明的保护内容不局限于以上实施例。在不背离本发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims (11)

  1. 一种适用于单细胞测序的猕猴桃根组织细胞核的制备方法，其特征在于，包括如下具体步骤：A method for preparing kiwifruit root tissue cell nuclei suitable for single-cell sequencing, characterized in that it comprises the following specific steps:
    1）配置KR裂解液：首先配置KR裂解液，并将配置好的溶液预冷；1) Prepare KR lysis buffer: First, prepare KR lysis buffer and pre-cool the prepared solution;
    2）破碎样本：将新鲜取材或者冻存的猕猴桃根组织样本转入离心管，加入预冷的所述KR裂解液及钢珠，依照仪器操作说明置于破碎仪上对组织进行破碎；2) Sample crushing: transfer freshly collected or frozen kiwifruit root tissue samples into a centrifuge tube, add the pre-cooled KR lysis solution and steel beads, and crush the tissue on a crusher according to the instrument operating instructions;
    3）裂解细胞：破碎后将离心管置于冰上孵育裂解；3) Lyse cells: After disruption, place the centrifuge tube on ice and incubate for lysis;
    4）筛网过滤：将裂解后的组织裂解液转入离心管中，加入预冷的PBS，冰上静置1分钟，吸出组织裂解液，加入滤网中进行过滤得到过滤液；4) Screen filtration: transfer the tissue lysate after lysis into a centrifuge tube, add pre-cooled PBS, let stand on ice for 1 minute, aspirate the tissue lysate, add it into the filter screen to filter and obtain the filtrate;
    5）筛网过滤：使用滤网对步骤4）中的过滤液再进行一次过滤；5) Screen filtration: Use the screen to filter the filtrate in step 4) again;
    6）离心收集沉淀：将步骤5）中的过滤液进行离心，丢弃上清，沉淀使用PBS进行重悬；6) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 5), discard the supernatant, and resuspend the precipitate in PBS;
    7）过滤：将步骤6）中的沉淀重悬液全部加入分选柱中进行过滤；7) Filtration: Add all the precipitate resuspension in step 6) into the separation column for filtration;
    8）离心收集沉淀：将步骤7）中的过滤液进行离心，丢弃上清，沉淀使用PBS进行重悬。8) Collect the precipitate by centrifugation: Centrifuge the filtrate in step 7), discard the supernatant, and resuspend the precipitate in PBS.
  2. 如权利要求1所述的制备方法，其特征在于，步骤（1）中，所述KR裂解液包含以下成份（终浓度）：2%~3% （v/v）L-酒石酸、15~25mM柠檬酸钠、0.2~0.4%（m/v）皂素、0.2~0.4U/mL 重组型RNase抑制剂。The preparation method as described in claim 1 is characterized in that in step (1), the KR lysate contains the following components (final concentration): 2%~3% (v/v) L-tartaric acid, 15~25mM sodium citrate, 0.2~0.4% (m/v) saponin, and 0.2~0.4U/mL recombinant RNase inhibitor.
  3. 如权利要求1所述的制备方法，其特征在于，步骤（2）中，所述破碎仪为万柏生物高通量组织破碎仪；所述破碎的条件为1200~1300rpm，170~180s；所述1ml的KR裂解液中添加50~100mg猕猴桃根组织。The preparation method according to claim 1, characterized in that in step (2), the disruptor is a Wanbo Bio high-throughput tissue disruptor; the disrupting conditions are 1200~1300rpm, 170~180s; and 50~100mg of kiwifruit root tissue is added to 1ml of KR lysis solution.
  4. 如权利要求1所述的制备方法，其特征在于，步骤（3）中，所述孵育的时间为7~8分钟。The preparation method according to claim 1, characterized in that in step (3), the incubation time is 7 to 8 minutes.
  5. 如权利要求1所述的制备方法，其特征在于，步骤（4）中，所述滤网为FALCON 40μm Cell Strainer，孔径40μm；所述过滤全程在0~5℃操作。The preparation method according to claim 1, characterized in that in step (4), the filter is a FALCON 40 μm Cell Strainer with a pore size of 40 μm; and the filtration is performed at 0-5°C throughout the entire process.
  6. 如权利要求1所述的制备方法，其特征在于，步骤（6）中，所述离心的温度为4-6℃，离心的时间为10-15分钟，离心力为300~500×g。The preparation method according to claim 1, characterized in that in step (6), the centrifugation temperature is 4-6°C, the centrifugation time is 10-15 minutes, and the centrifugal force is 300-500×g.
  7. 如权利要求1所述的制备方法，其特征在于，步骤（7）中，所述分选柱为Miltenyi MS Column；所述过滤，全程在0~5℃操作。The preparation method according to claim 1, characterized in that in step (7), the separation column is a Miltenyi MS Column; and the filtration is performed at 0-5°C throughout the process.
  8. 如权利要求1所述的制备方法，其特征在于，步骤（8）中，所述离心的温度为4-6℃，离心的时间为10-15分钟，离心力为300~500×g。The preparation method according to claim 1, characterized in that in step (8), the centrifugation temperature is 4-6°C, the centrifugation time is 10-15 minutes, and the centrifugal force is 300-500×g.
  9. 一种KR裂解液，其特征在于，所述KR裂解液包含以下成份（终浓度）：2%~3% （v/v）L-酒石酸、15~25mM柠檬酸钠、0.2~0.4%（m/v）皂素、0.2~0.4U/mL 重组型RNase抑制剂。A KR lysis buffer, characterized in that the KR lysis buffer contains the following components (final concentration): 2%~3% (v/v) L-tartaric acid, 15~25mM sodium citrate, 0.2~0.4% (m/v) saponin, and 0.2~0.4U/mL recombinant RNase inhibitor.
  10. 一种试剂/试剂盒，其特征在于，其包含如权利要求9所述的KR裂解液。A reagent/kit, characterized in that it comprises the KR lysate as described in claim 9.
  11. 如权利要求9所述的KR裂解液或如权利要求10所述的试剂/试剂盒在制备适用于单细胞测序的猕猴桃根组织细胞核的方法、猕猴桃根组织细胞核转录组测序、猕猴桃根组织细胞核ATAC测序研究中的应用。The use of the KR lysate as described in claim 9 or the reagent/kit as described in claim 10 in a method for preparing kiwifruit root tissue nuclei suitable for single-cell sequencing, kiwifruit root tissue nuclei transcriptome sequencing, and kiwifruit root tissue nuclei ATAC sequencing research.
PCT/CN2023/090396 2022-09-28 2023-04-24 Method for preparing plant cell nucleus suitable for single-cell sequencing of chinese actinidia root tissue and optimizing suspension WO2024066317A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211189716.1A CN115612720A (en) 2022-09-28 2022-09-28 Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing
CN202211189716.1 2022-09-28

Publications (1)

Publication Number Publication Date
WO2024066317A1 true WO2024066317A1 (en) 2024-04-04

Family

ID=84860642

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/090396 WO2024066317A1 (en) 2022-09-28 2023-04-24 Method for preparing plant cell nucleus suitable for single-cell sequencing of chinese actinidia root tissue and optimizing suspension

Country Status (2)

Country Link
CN (1) CN115612720A (en)
WO (1) WO2024066317A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115612720A (en) * 2022-09-28 2023-01-17 上海欧易生物医学科技有限公司 Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110136220A1 (en) * 2009-12-07 2011-06-09 Takako Nagata Method for Isolating Nuclei
US20190338279A1 (en) * 2016-12-07 2019-11-07 Mgi Tech Co., Ltd. Method for constructing single cell sequencing library and use thereof
CN113444678A (en) * 2020-03-26 2021-09-28 武汉华大医学检验所有限公司 Method for preparing single cell nuclear suspension, single cell sequencing method and kit
CN114277094A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Lysate for extracting plant cell nucleus
CN114277093A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Method for extracting plant cell nucleus
CN114350747A (en) * 2021-12-22 2022-04-15 上海欧易生物医学科技有限公司 Method for simultaneously separating exosome and cell nucleus and method for simultaneously sequencing exosome and single cell nucleus
CN115074310A (en) * 2022-06-24 2022-09-20 上海欧易生物医学科技有限公司 Method for simultaneously preparing single cell suspension and single cell nuclear suspension based on same lung tissue sample and application
CN115612720A (en) * 2022-09-28 2023-01-17 上海欧易生物医学科技有限公司 Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110136220A1 (en) * 2009-12-07 2011-06-09 Takako Nagata Method for Isolating Nuclei
US20190338279A1 (en) * 2016-12-07 2019-11-07 Mgi Tech Co., Ltd. Method for constructing single cell sequencing library and use thereof
CN113444678A (en) * 2020-03-26 2021-09-28 武汉华大医学检验所有限公司 Method for preparing single cell nuclear suspension, single cell sequencing method and kit
CN114350747A (en) * 2021-12-22 2022-04-15 上海欧易生物医学科技有限公司 Method for simultaneously separating exosome and cell nucleus and method for simultaneously sequencing exosome and single cell nucleus
CN114277094A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Lysate for extracting plant cell nucleus
CN114277093A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Method for extracting plant cell nucleus
CN115074310A (en) * 2022-06-24 2022-09-20 上海欧易生物医学科技有限公司 Method for simultaneously preparing single cell suspension and single cell nuclear suspension based on same lung tissue sample and application
CN115612720A (en) * 2022-09-28 2023-01-17 上海欧易生物医学科技有限公司 Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王权 等 (WANG, QUAN ET AL.): "单细胞测序的技术概述 (Overview of the Technology of Single Cell Sequencing)", 中国医药导刊 (CHINESE JOURNAL OF MEDICINAL GUIDE), vol. 22, no. 7, 31 December 2020 (2020-12-31), pages 433 - 439, XP055894854 *

Also Published As

Publication number Publication date
CN115612720A (en) 2023-01-17

Similar Documents

Publication Publication Date Title
JP4554080B2 (en) Methods and reagents for storing RNA in cell and tissue samples
WO2024066317A1 (en) Method for preparing plant cell nucleus suitable for single-cell sequencing of chinese actinidia root tissue and optimizing suspension
WO2018006782A1 (en) Methods for isolating different types of single cells from ovary
EP1654387B1 (en) Materials and methods for capture of pathogens and removal of aurintricarboxylic acid from a sample
JP2011522529A (en) Method for isolating short-chain nucleic acids
CA2310782A1 (en) Methods of nucleic acid isolation
US20220244148A1 (en) Composition and Method for Disrupting Tissue Material
CN102137931A (en) Method for filtering nucleic acids, in particular from fixed tissue
EP3783113B1 (en) Isolation of high molecular weight dna using beads
CN112538475A (en) Extraction method, sequencing method and kit of plant genome DNA
CN112280828A (en) In vitro tissue cell nucleus separation method for reducing single cell amplification bias
Rickwood et al. Isolation and characterization of nuclei and nuclear subfractions
CN114277093A (en) Method for extracting plant cell nucleus
EP4215618A1 (en) Method for analyzing cell epigenomics from multiple dimensions
Cerritos-Castro et al. Method for CaOx crystals isolation from plant leaves
CN114015752B (en) Mouse retina frozen tissue cell nucleus suspension suitable for single cell sequencing and preparation method and application thereof
CN116478917A (en) Animal adipose tissue cell nucleus extraction kit and preparation method and application thereof
GB2584562A (en) Purification method
WO2023246266A1 (en) Method for simultaneously preparing single cell suspension and single cell nucleus suspension based on same tissue sample, and use
JP2013039134A (en) Material and method for capture of pathogen and removal of aurintricarboxylic acid from sample
CN114350610B (en) Kit for separating brain and spinal cord tissue cell nuclei and cell nucleus separation method
EP3985112A1 (en) Purification method
CN117987510A (en) High-quality corn root tip cell nucleus separation method for single cell sequencing
CN114410744A (en) Method for processing sample, nucleic acid extraction method and library thereof
CN117866871A (en) Method for extracting cell nucleus from plant leaves suitable for ATAC-seq and application

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23869573

Country of ref document: EP

Kind code of ref document: A1