CN115612720A

CN115612720A - Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing

Info

Publication number: CN115612720A
Application number: CN202211189716.1A
Authority: CN
Inventors: 范黎明; 朱燕敏; 佟思雨; 王佳琦; 殷昊; 肖云平
Original assignee: Shanghai Oe Biotech Co ltd
Current assignee: Shanghai Oe Biotech Co ltd
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2023-01-17
Also published as: WO2024066317A1

Abstract

The invention discloses a preparation method of kiwi root tissue cell nucleus suitable for single cell sequencing, which is prepared by an original lysate formula and a cell nucleus suspension optimization method to obtain cell nucleus with high quality. Compared with the lysate formula with 6-7 components in the prior art, the lysate formula is obviously reduced; the preparation of the cell nucleus can be completed within 30 minutes by the experimental process. The method disclosed by the invention avoids the steps of sucrose deposition and the like, saves reagents and consumables, reduces the cost, greatly shortens the time, improves the efficiency and is convenient for developing a large number of sample experiments. The invention also discloses KR lysate, a reagent/a kit and application thereof in a method for preparing kiwi root tissue cell nucleuses suitable for single cell sequencing.

Description

Plant cell nucleus preparation and suspension optimization method suitable for kiwi root tissue single cell sequencing

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of plant cell nucleuses, in particular to a preparation method of kiwi root tissue cell nucleuses, and application and a use method thereof in plant single cell sequencing.

Background

The single cell sequencing technology analyzes the molecular mechanism behind the life phenomenon by 'single cell resolution' through measuring the transcriptome of a single cell, is a top technical means of life science research, and is widely applied. However, due to the existence of cell wall, plant tissue is "difficult and serious" to sequence single cells, and the plant cells must be dissociated into protoplasts first to sequence the single cells. The main components of the cell wall are polysaccharides such as cellulose, and these macromolecules have variable structures and stable chemical properties, and are one of the most difficult-to-degrade substances. Therefore, at present, most plant tissues are difficult to prepare protoplasts meeting the single cell sequencing requirement, and the research of single cell sequencing of plants is greatly limited. In addition, the preparation of protoplasts causes great changes to plant cells, which can induce transcriptional changes, resulting in serious decrease in the reliability of sequencing results, and data that cannot reflect real biological changes.

In order to avoid the problems of difficulty in separating cells from tissues, data distortion caused by the dissociation process and the like, the animal tissues can be tested by adopting single cell nucleus sequencing, and a very good effect is achieved. Therefore, the method of sequencing single cells of plants by preparing plant cell nuclei by using animal tissues is an effective means for breaking through the current technical problems. However, the current methods for preparing cell nuclei from plant tissues are all applied to the related research of arabidopsis thaliana, and are not suitable for the research of kiwi root tissues, and after the kiwi root tissues are used for preparing the cell nuclei, indexes such as the number of the cell nuclei and the proportion of cell fragments cannot meet the requirement of single cell sequencing, so that the development of a novel method for preparing the cell nuclei, which is suitable for the research of single cell sequencing of kiwi root tissues, is urgently needed.

Disclosure of Invention

Although arabidopsis thaliana has a mature technical scheme for preparing Cell Nuclei, for example, in the experimental steps described in Identification of Open chromosome Regions in Plant Genomes Using ATAC-Seq and Isolation of Plant Root nucleic acid for Single Cell RNA Sequencing, the former is mainly applied to genome Sequencing research of arabidopsis thaliana, and the latter is mainly applied to transcriptome Sequencing research, and the two methods can respectively prepare Cell nucleus suspensions of arabidopsis thaliana leaf tissues and Root tissues. The kiwi fruit is a large-scale fallen leaf vine, has extremely low tissue similarity with arabidopsis, has larger component difference of cell walls, and has larger difference of tissue and cell components due to different growth environments. The method for preparing the cell nucleus by using arabidopsis cannot prepare the suspension of the cell nucleus of the kiwi root tissue which meets the single cell sequencing.

Aiming at the blank and the defects in the prior art, the invention provides a preparation method of kiwi fruit root tissue cell nucleus suitable for single cell sequencing, and a corresponding lysate formula and a cell nucleus suspension optimization method on the basis of a large amount of intensive research and experiments (wherein, the cell nucleus suspension optimization method mainly optimizes the contents of removing calcium oxalate crystals deposited in kiwi fruit root tissues/cells, removing mRNA released into suspension in the process of extracting the kiwi fruit root tissues and removing tissue fragments in the cell nucleus suspension).

The preparation method of the kiwi fruit root tissue cell nucleus suitable for single cell sequencing provided by the invention comprises the following specific steps:

1) Preparing KR lysate: firstly, KR lysate is prepared, and the prepared solution is precooled.

2) Crushing a sample: and (3) transferring the fresh kiwi fruit root tissue sample into a centrifuge tube, adding the precooled KR lysate and steel balls, and placing the sample on a crusher according to the instrument operation instruction to crush the tissue.

3) Cell lysis: after disruption, the tubes were incubated on ice for lysis.

4) Filtering with a screen: transferring the tissue lysate after the lysis in the step 3) into a centrifuge tube, and adding precooled PBS. The mixture was allowed to stand on ice for 1 minute. The tissue lysate is aspirated and filtered in a filter screen.

5) Filtering with a screen: filtering the filtrate obtained in the step 4) again by using a filter screen.

6) And (3) centrifuging and collecting precipitates: centrifuging the filtrate obtained in step 5). The supernatant was discarded and the pellet resuspended with PBS.

7) And (3) filtering: and (3) adding all the precipitate resuspension in the step 6) into a sorting column for filtering.

8) And (3) centrifuging and collecting precipitates: the filtrate from step 7) was centrifuged, the supernatant discarded and the pellet resuspended using 100. Mu.L PBS.

In the step (1), the KR lysis solution contains the following components (final concentration):

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), 0.2-0.4U/mL RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613); preferably 3% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613).

In the step (1), L-tartaric acid is commonly used as an acid agent of beverages and other foods, and is used for neutralizing cell contents released after tissues and cells are crushed, stabilizing the PH of a lysate and keeping cell nuclei complete; meanwhile, L-tartaric acid is used as a reducing agent in the lysate and plays a role in keeping the activity of RNase inhibitor.

In the step (1), the saponin is mainly used as a medical raw material and is also used as an antiseptic material and an emulsifier component, and in the invention, the saponin is used as a component of the lysis solution to perform the functions of lysing cell membranes and releasing cell nuclei.

In the step (1), 2-3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) is added when KR lysate is prepared, the solution is fully mixed and placed on ice for precooling, and then 0.2-0.4U/mL RNase inhibitor is added. The purpose is that 2-3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) can keep the activity of 0.2-0.4U/mL RNase inhibitor.

In the step (1), the precooling temperature is 0-5 ℃; preferably, the temperature is pre-cooled by placing in a refrigerator at 4 ℃.

In the step (2), the centrifugal tube is

2.0mL MaxyClear Snaplock Microcentrifuge Tube, RNase-/DNase-free and nonpyrogenic (Axygen, MCT-200-C) (Aijin 2.0ml nuclease-free transparent centrifuge tubes).

In the step (2), a disruptor is usually used for nucleic acid extraction, and the action in the invention is to fully destroy plant tissues through steel balls and assist KR lysate to complete the preparation of crude cell nucleus suspension.

In the step (2), the diameter of the steel ball is 5mm.

In the step (2), the crusher is a Wanbai biological high-throughput tissue crusher (onebaio-48 p).

In the step (2), the crushing conditions are 1200-1300rpm and 170-180 s; preferably, 1200rpm,180s.

In the step (2), the kiwi fruit root can refer to the root tip tissue of any variety of kiwi fruit, and comprises a meristem region, an elongation region and a mature region.

In the step (2), the fresh weight of the kiwi fruit root tissue is 50-100 mg; preferably, it is 100mg.

In step (3), the incubation is performed to remove structures such as cell membranes outside the cell nucleus by using KR lysate at a temperature that maintains the integrity of messenger RNA (mRNA) in the cell nucleus.

In the step (3), the incubation time is 7-8 minutes; preferably, it is 7min.

In the step (4), the filter screen is FALCON 40 μm Cell Strainer (FALCON, 352340), and the aperture is 40 μm.

In step (4), the PBS was Gibco PBS pH7.4 (1 ×) (Gibco, 10010-031).

In step (4), the centrifuge tube is a Corning Centristar 15mL centrifuge tube (Corning, 430790).

In the step (4), the whole filtering process is operated at 0-5 ℃; preferably, for operations on ice.

Standing for 1 minute on ice in the step (4) aims to enable large tissue blocks to settle to the bottom of a centrifuge tube, and the tissue blocks do not settle completely due to too short standing time, so that the screen mesh is blocked during screen mesh filtration, and cell nucleus loss is caused; too long a standing time poses a risk of mRNA degradation, which will directly lead to failure of the experiment. The time for the tissue mass to settle at 1min ensures complete settling and no risk of mRNA degradation.

In the step (5), the filter screen is FALCON 40 μm Cell Strainer (FALCON, 352340), and the aperture is 40 μm.

In the step (6), the centrifugation temperature is 4-6 ℃; preferably, it is 4 ℃.

In the step (6), the centrifugation time is 10-15 minutes; preferably, it is 10 minutes.

In the step (6), the centrifugal force of the centrifugation is 300 to 500 Xg, preferably 300 Xg.

In the step (6), the PBS is pre-cooled in a refrigerator at 4 ℃ 30min in advance.

In step (7), the sorting Column is Miltenyi MS Column (Miltenyi, 130-042-201).

In the step (7), the sorting column is usually used for sorting animal specific cell types by combining an antibody with magnetic beads with a specific antigen on the surface of a target cell membrane under a magnetic field, and in the invention, by utilizing the needle-shaped morphological characteristics of calcium oxalate crystals and the stacking characteristics of nano materials in the sorting column under a non-magnetic field, cell nuclei with small volume can pass through the sorting column along with a liquid flow, and the needle-shaped calcium oxalate crystals can be left in the sorting column and cannot pass through along with the liquid flow, so that the aim of purifying the cell nuclei is fulfilled.

In the step (7), the whole filtering process is operated at 0-5 ℃; preferably, for operations on ice.

In the step (8), the centrifugation temperature is 4-6 ℃; preferably, it is 4 ℃.

In the step (8), the centrifugation time is 10-15 minutes; preferably, it is 10 minutes.

In the step (8), the centrifugal force of the centrifugation is 300-500 Xg; preferably, it is 300 Xg.

In the step (8), the PBS is precooled on ice.

The invention also provides the kiwi fruit root tissue cell nucleus obtained by the method and suitable for single cell sequencing.

The invention also provides application of the kiwi fruit root tissue cell nucleus in single cell sequencing.

In a specific embodiment, the method of the present invention comprises the following specific steps:

1) KR lysate was prepared.

The lysate was prepared according to the following ingredients:

2-3% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.2-0.4U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613).

And (3) placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

Fresh kiwi root tissue samples or frozen kiwi root tissue samples are transferred into 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube is placed on a clamping plate for crushing and a spanner is tightened, the rotating speed of the instrument is adjusted to 1200-1300rpm, and 170-180 seconds are carried out. The instrument was started for tissue disruption.

3) The cells were lysed.

After the instrument was completely stopped, the centrifuge tube was removed and inserted into ice, and incubated for 7 minutes.

4) And (5) filtering.

The tissue lysate from step (3) was transferred to a new 15mL centrifuge tube (Corning, 430790) using a pipette. 9mL of ice-cold PBS (Gibco, 10010-031) were added to the centrifuge tube. The mixture was allowed to stand on ice for 1 minute.

The lysate was pipetted out successively and filtered through a 40 μm sieve (FALCON, 352340). The filtrate was collected in a new 50mL centrifuge tube (Corning, 430828).

5) And (4) filtering.

The filtrate obtained in step (4) was gradually aspirated by a pipette, and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected into a new 15mL centrifuge tube.

6) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (5) in a centrifuge, and centrifuging at 300 Xg for 10 minutes at 4 ℃.

After centrifugation was complete, the tube was carefully removed, the supernatant carefully decanted, and the pellet resuspended in 3mL ice-cold PBS.

7) And (4) filtering.

The 3mL of the resuspension from step (6) was added to the MS Column (Miltenyi, 130-042-201) and the filtrate was collected in a new 15mL centrifuge tube and filtered by resting on ice until the liquid was completely filtered into the 15mL centrifuge tube.

8) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (7) in a centrifuge, and centrifuging at 300 Xg for 10 minutes at 4 ℃.

After centrifugation, the tubes were carefully removed, the supernatant carefully decanted, and the pellet resuspended in 100. Mu.L of ice-precooled PBS

9) Counting by microscopic examination.

Take 5. Mu.L of the resuspended fluid from step (8) and mix it with DAPI dye 1:1. Total nuclei and concentration were counted using a hemocytometer.

10 ) single cell sequencing machine.

Single cell sequencing was performed on the machine according to the 10 Xgenomics kit instructions.

The invention also provides KR lysate which comprises the following components (final concentration): 2-3% (v/v) L-tartaric acid, 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin and 0.2-0.4U/mL recombinant RNase inhibitor.

The invention also provides a reagent/kit, which is characterized by comprising the KR lysate as described above.

The invention also provides an application of the KR lysate or the reagent/kit in preparation of a method for sequencing a kiwi root tissue cell nucleus suitable for single cell sequencing, sequencing kiwi root tissue cell nucleus transcriptome and sequencing kiwi root tissue cell nucleus ATAC.

Compared with the prior art, the invention has the following beneficial effects:

firstly, the current technical blank is filled: at present, a preparation method of kiwi root tissue cell nuclei suitable for a single cell sequencing experiment is lacked, and the application of a single cell sequencing technology in kiwi root tissue research is seriously hindered. The technical scheme provided by the invention solves the problem, and can powerfully promote the development and wide application of single cell sequencing research of kiwi root tissues.

Secondly, the quality of the Chinese gooseberry root cell nucleus suspension is improved. The kiwi root tissue contains a large amount of impurities, polysaccharide and the like, a large amount of impurities such as fragments, intracellular crystals and the like exist in the cracked suspension, the quality of the kiwi root tissue can not meet the requirements of 10x Genomics on machine, and the research progress of the kiwi root tissue is seriously hindered. The quality of the cell nucleus prepared according to the technical scheme of the invention is high (the quality of the sample is 3 multiplied by 10 from the number of the cell nucleus) ⁵ The fragment rate is more than 70 percent and is improved to 2.26 multiplied by 10 ⁶ ～4.12×10 ⁶ The fragment rate is 3-5%. ) And impurities such as cell fragments, intracellular crystals and the like are basically and completely removed, so that the subsequent single cell experiment can be smoothly carried out.

Thirdly, the cost is low and the time consumption is short. The technical scheme provided by the invention only uses 4 components of the cracking solution, and compared with 6-7 components in the prior art, the components are obviously reduced; the preparation of the cell nucleus can be completed within 30 minutes by the experimental process. The method avoids the steps of sucrose deposition and the like, saves reagents and consumables, reduces the cost, greatly shortens the time, improves the efficiency and is convenient for developing a large number of sample experiments.

Drawings

FIG. 1 is a microscopic view of cell nuclei. The figure shows the cell nucleus prepared by the technical method of the invention. It can be seen that the impurities such as cell debris and calcium oxalate crystals are completely removed, and only the impurities smaller than the cell nucleus exist in a very low proportion.

FIG. 2 shows the results of quality control of single-cell transcriptome-sequenced cDNA according to examples 1 to 4 of the present invention (Agilent 4150 bioanalyzer).

FIG. 3 is a graphic representation of needle crystals of calcium oxalate in tissue.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the concept of the invention. All falling within the scope of the present invention.

Example 1

1) And preparing a lysis solution. The lysate was prepared according to the following ingredients:

2% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613). And placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube was placed on a jaw for crushing and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 170 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

The filtrate obtained in step (4) was gradually aspirated by a pipette and filtered through a 40 μm filter (FALCON, 352340). The filtrate was collected into a new 15mL centrifuge tube.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

After centrifugation was complete, the tube was carefully removed, the supernatant carefully decanted, and the pellet resuspended in 10mL ice-precooled PBS

9) Washing the precipitate

Placing the centrifuge tube in the step (8) in a centrifuge, and centrifuging at 300 Xg for 10 minutes at 4 ℃.

After centrifugation was complete, the tubes were carefully removed, the supernatant carefully decanted, and the pellet resuspended in 100. Mu.L of ice-precooled PBS.

10 Quality inspection, microscopic counting.

The supernatant was aspirated and the concentration of RNA in the suspension was measured using a NanoDrop microspectrophotometer and a fluorospectrophotometer.

Calculating the concentration of viable cell nuclei, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: taking 5 mu L of the heavy suspension liquid in the step (9), mixing the heavy suspension liquid with the DAPI dye solution 1:1 uniformly, sucking 10ul of the mixed solution by a liquid transfer machine, adding the mixed solution into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

11 Single cell sequencing machine.

According to the instructions of 10 Xgenomics: chromiumNext GEM Single Cell 3' reagent Kit v3.1, CG000204REV C, were subjected to Single Cell sequencing procedures.

Results and analysis: the results are shown in Table 1; the proportion of living cells, the proportion of cell aggregates, the proportion of fragments and the cell concentration all reach the single cell sequencing requirement of 10 Xgenomics company (Table 1, figure 1) and the cDNA length is normal (figure 2). The library construction result shows that the sequencing effect of extracting the cell nucleus suspension from the frozen sample to carry out the single cell transcriptome is good.

Example 2

1) And (4) preparing a lysis solution. The lysate was prepared according to the following ingredients:

3% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613). And (3) placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1300rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

After the instrument was completely stopped, the centrifuge tube was removed and inserted into ice, and incubated for 8 minutes.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (5) in a centrifuge, and centrifuging at 500 Xg for 10 minutes at 4 ℃.

7) And (4) filtering.

8) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (7) in a centrifuge, and centrifuging at 500 Xg for 10 minutes at 4 ℃.

9) Washing the precipitate

Placing the centrifuge tube in the step (8) in a centrifuge, and centrifuging at 500 Xg for 10 minutes at 4 ℃.

10 Quality inspection, microscopic counting.

11 ) single cell sequencing machine.

According to the 10 Xgenomics instructions: chromiumNext GEM Single Cell 3' reagent Kit v3.1, CG000204REV C, for Single Cell sequencing operations.

Results and analysis: the results are shown in Table 1; the RNA content in the suspension is extremely low; the live cell proportion, the cell agglomeration proportion, the fragment proportion and the cell concentration can all reach the single cell sequencing requirement of 10 Xgenomics company, the cDNA length is normal, and the library construction result shows that the single cell transcriptome sequencing effect of extracting the cell nucleus suspension from the frozen sample is good (figure 2).

Example 3

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 170 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (4) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

9) Washing the precipitate

10 Quality inspection, microscopic counting.

11 ) single cell sequencing machine.

Example 4

3% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 25mM sodium citrate, 0.4% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.4U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613). And placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

100mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). And (2) adding 1mL of the lysis solution precooled on ice in the step (1), and adding a steel ball for crushing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1300rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

After centrifugation was complete, the centrifuge tube was carefully removed, the supernatant carefully decanted, and the pellet resuspended using 3mL of ice-cold PBS.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

9) Washing the precipitate

10 Quality inspection, microscopic counting.

11 ) single cell sequencing machine.

According to the 10 Xgenomics instructions: chromiumNext GEM Single Cell 3' reagent Kit v3.1, CG000204REV C, were subjected to Single Cell sequencing procedures.

Results and analysis: the results are shown in Table 1; the proportion of living cells, the proportion of cell aggregates, the proportion of fragments and the cell concentration all reach the single cell sequencing requirement of 10 Xgenomics company (Table 1, figure 1) and the cDNA length is normal (figure 2). The library construction result shows that the single cell transcriptome sequencing effect of extracting the cell nucleus suspension from the frozen sample is good.

Comparative example 1

This example performs Arabidopsis thaliana leaf cell nucleus preparation according to the Identification of Open chromosome Regions in Plant Genomes Using ATAC-Seq.

1) And preparing a lysis solution.

The cell nucleus purification buffer was prepared according to the following composition

20mM MOPS pH 7,40mM NaCl,90mM KCl,2mM EDTA,0.5mM EGTA,0.5mM spermidine,0.2mM spermine,and 1×Roche Complete protease inhibitors.

Preparing a cell nucleus extracting solution 1 according to the following components:

0.25M Sucrose,10mM Tris–HCl pH 8,10mM MgCl2,1％Triton X-100,and 1×Roche Complete Protease Inhibitors.

preparing a cell nucleus extracting solution 2 according to the following components:

1.7M Sucrose,10mM Tris–HCl pH 8,2mM MgCl2,and 0.15％Triton X-100,1×Roche Complete Protease Inhibitors.

and placing the prepared solution on ice for precooling.

2) And (4) crushing the sample.

50mg of Arabidopsis thaliana leaf tissue sample is transferred into a mortar, and a certain amount of liquid nitrogen is poured into the mortar for grinding until the tissue is completely changed into powder. During which time the addition of liquid nitrogen was noted to maintain low temperatures.

3) The cells were lysed.

The milled powder was transferred to a new mortar containing 10mL of ice-precooled nuclear purification buffer. The powder was resuspended with stirring using a pestle.

4) And (5) filtering.

The tissue lysate obtained in step (3) is gradually aspirated by a pipette, filtered through a 70 μm filter screen, and the filtrate is collected in a new 15mL centrifuge tube. Note that the centrifuge tubes should be inserted in ice.

5) The precipitate was collected by centrifugation.

Centrifuging the centrifuge tube in step (4) at 1200 Xg and 4 ℃ for 10 minutes in a centrifuge.

The tube was carefully removed and the supernatant was aspirated off and discarded.

6) The precipitate was collected by centrifugation.

Resuspend the nuclear pellet in step (5) using 1mL of pre-cooled nuclear extract 1.

The resuspension was aspirated and added to a fresh 1.5mL centrifuge tube. The tube was placed in a centrifuge and centrifuged at 12000 Xg at 4 ℃ for 10 minutes.

7) The precipitate was collected by centrifugation.

To a new 1.5mL centrifuge tube was added 300. Mu.L of cell nucleus extract 2.

Resuspend the nuclear pellet from step (6) using 300 μ L of pre-cooled nuclear extract 2. The resuspension solution was carefully added to the centrifuge tube containing the cell nucleus extract 2 to allow the solution to stratify.

The tube was placed in a centrifuge and centrifuged at 16000 Xg at 4 ℃ for 10 minutes.

The pellet was resuspended using 1mL of pre-cooled nuclear purification buffer.

8) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: taking 5 mu L of the heavy suspension liquid in the step (8), mixing the heavy suspension liquid with the DAPI dye solution 1:1 uniformly, sucking 10ul of the mixed solution by a liquid transfer machine, adding the mixed solution into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

9) Results and analysis

The results showed that the number of nuclei was 2X 10 ⁶ The proportion of the debris impurities is 28 percent. Meets the minimum requirements of single-cell transcriptome sequencing on the sample.

Comparative example 2

This example performs kiwi leaf tissue cell nucleus preparation according to the Identification of Open chromosome Regions in Plant Genomes Using ATAC-Seq. The remaining operation steps were the same as in comparative example 1.

The results are shown in Table 2.

Comparative example 3

This example performs actinidia root tissue cell nucleus preparation according to the Identification of Open chromosome Regions in Plant Genomes Using ATAC-Seq. The remaining operation steps were the same as in comparative example 1.

The results are shown in Table 2.

Comparative example 4

This example follows the Plant tissue Cell nucleus extraction kit (CelLytic) according to the Isolation of Plant Root nucleus for Single Cell RNA Sequencing ^TM PN isolation/extraction kit, sigma, code CELLYTPN1-1 KT) for root tissue and cell nuclei preparation.

1) Preparation of lysate

500 μ L of the IB lysate was poured into fresh, sterile and pre-chilled 60mm petri dishes.

2) Taking materials

Roots of 7-day-old arabidopsis plants were cut with a double-sided razor, and fresh roots were collected.

3) Transfer material

Fresh root samples were transferred to 60mm petri dishes with forceps and the roots were all immersed in the NIB lysate.

4) Tissue mincing

The root specimens taken were chopped with a double-sided razor blade for about 5 minutes.

5) Tissue lysis

The lysis was performed by incubating for 15 minutes at 4 ℃ with gentle horizontal shaking.

6) Filtration

Under low temperature environment, a 40 μm filter is wetted by 500 μ L NIB, and then the nuclear suspension after root lysis is sucked by a disposable large-caliber suction head, and is slowly filtered, and then the filter is washed by 500 μ L NIB to collect the residual cell nuclei.

7) Screen filtration

The filtered nuclear suspension was then filtered twice using a 30 μm filter using the method of step 7, and the filter was washed with 500 μ l of ib, collecting the remaining nuclei and removing debris.

8) Microscopic examination counting

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) taking 5 mu L of the heavy suspension liquid obtained in the step (8), uniformly mixing the heavy suspension liquid with the DAPI dye solution 1:1, sucking 10uL of the mixed solution by a liquid transfer device, adding the mixed solution into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 2

Comparative example 5

This example follows the Plant tissue Cell nucleus extraction kit (CelLytic) according to the Isolation of Plant Root nucleus for Single Cell RNA Sequencing ^TM PN separation/extraction kit, sigma, code CELLYTPN1-1 KT) for kiwi leaf tissue cell nuclei preparation. The remaining operation steps were the same as in comparative example 4.

The results are shown in Table 2.

Comparative example 6

This example follows the Plant tissue Cell nucleus extraction kit (CelLytic) according to the Isolation of Plant Root nucleus for Single Cell RNA Sequencing ^TM PN separation/extraction kit, sigma, code CELLYTPN1-1 KT) for preparing the kiwi root tissue cell nucleus. The remaining operation steps were the same as in comparative example 4.

The results are shown in Table 2.

Comparative example 7

This example uses ST1 buffer for actinidia root tissue cell nucleus preparation.

1) ST1 buffer was prepared. The buffer was prepared according to the following ingredients:

2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023). The prepared buffer solution was placed on ice for precooling.

2) And (4) crushing the sample.

Transferring 50mg of the kiwi fruit root tissue sample into a mortar, pouring a certain amount of liquid nitrogen, and grinding until the tissue is completely changed into powder. During which time the addition of liquid nitrogen was noted to maintain low temperatures.

3) The cells were lysed.

The milled powder was transferred to a fresh 15mL centrifuge tube containing 10mL of ice-pre-chilled buffer. The suspension was resuspended by inverting the top and bottom 15 times.

4) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (4) taking 5 mu L of the cell nucleus suspension obtained in the step (3), uniformly mixing the cell nucleus suspension with the DAPI staining solution 1:1, sucking 10ul of the mixture by a liquid transfer device, adding the mixture into a sample adding hole of a blood counting plate, counting, and observing the number of the cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

5) And (5) measuring parameters.

The buffer pH in step (3) was measured using a pH meter (METTLER TOLEDO FE28-CN, 30595013).

The results are shown in Table 2.

Comparative example 8

This example uses ST2 buffer composition of 15mM sodium citrate for the preparation of kiwi root tissue nuclei, and the remaining procedure is the same as in comparative example 7.

The results are shown in Table 2.

Comparative example 9

This example uses ST3 buffer components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 20mM Tris-HCl (pH = 7.5) for the preparation of actinidia root tissue nuclei, and the remaining procedures are the same as in comparative example 7.

Comparative example 10

This example uses ST4 buffer with 15mM sodium citrate, 20mM MOPS (3- (N-morpholino) propanesulfonic acid, sigma-Aldrich, 1132-61-2) for the preparation of actinidia root tissue nuclei, and the remaining procedures were the same as in comparative example 7.

The results are shown in Table 2.

Comparative example 11

This example uses ST buffer solution components of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate for the preparation of kiwi root tissue nuclei, and the rest of the procedure is the same as in comparative example 7.

The results are shown in Table 2.

Comparative example 12

This example was carried out using ST5 buffer containing 1.5% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 10mM sodium citrate to prepare the nuclei of tissue of kiwi fruit root, and the rest of the procedure was the same as in comparative example 7.

The results are shown in Table 2.

Comparative example 13

This example uses ST6 buffer solution composition of 4% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 30mM sodium citrate for the preparation of kiwi root tissue nuclei, and the rest of the procedure is the same as in comparative example 7.

The results are shown in Table 2.

Comparative example 14

This example follows the preparation of actinidia root tissue nuclei using KR1 lysate.

1) KR1 lysate was prepared. The lysate was prepared 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). And placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). And (3) adding 1mL of the lysate precooled on ice in the step (1), and shearing the kiwi fruit roots into the lysate by using sterilizing scissors.

3) The cells were lysed.

The centrifuge tubes were inserted into ice and incubated for 8 minutes at rest.

4) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) taking 5 mu L of lysate in the step (3), uniformly mixing the lysate with the DAPI stain 1:1, sucking 10ul of lysate by using a pipette, adding the lysate into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by using a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 2.

Comparative example 15

This example followed the preparation of actinidia root tissue nuclei using KR2 lysate fraction of 2% (v/v) L-tartaric acid (nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) sodium deoxycholate (Sigma, 302-95-4), with the remaining procedure being the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 16

This example followed the preparation of actinidia root tissue nuclei using KR3 lysate fraction of 2% (v/v) L-tartaric acid (nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) digitonin (Sigma, 11024-24-1), with the remaining procedure being the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 17

This example followed the preparation of actinidia root tissue nuclei using KR4 lysate components of 2% (v/v) L-tartaric acid (nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (v/v) Triton X-100 (4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol, sigma, 9036-19-5), and the rest of the procedure was the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 18

This example followed the preparation of actinidia root tissue nuclei using KR5 lysate composition of 2% (v/v) L-tartaric acid (nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (nanking Reagent, 8047-15-2), with the remaining procedure being the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 19

This example followed the preparation of tissue nuclei of actinidia root according to the KR6 lysate composition of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.1% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the rest of the procedure was the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 20

This example followed the preparation of tissue nuclei of actinidia root according to the KR7 lysate composition of 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), and the rest of the procedure was the same as in comparative example 14.

The results are shown in Table 2.

Comparative example 21

This example followed the use of double-sided blades to chop kiwi root tissue, KR5 lysate lysis.

1) KR5 lysate was prepared. The lysate was prepared 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-cooling the prepared lysate on ice

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were placed in petri dishes on ice, and the roots were cut into 0.5mm pieces using a double-sided razor blade and transferred into 2mL centrifuge tubes (Axygen, MCT-200-C). 1mL of the lysate precooled on ice in step (1) was added.

3) The cells were lysed.

4) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (4) taking 5 mu L of lysate obtained in the step (3), uniformly mixing the lysate with the DAPI stain 1:1, sucking 10ul of lysate by using a liquid transfer device, adding the lysate into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by using a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 3.

Comparative example 22

This example followed the cutting of kiwi root tissue with scissors and the lysis of KR5 lysate, which was performed in the same manner as in comparative example 17.

The results are shown in Table 3.

Comparative example 23

This example followed the disruption of the kiwi root tissue using a glass homogenizer to homogenize, KR5 lysate was lysed.

1) KR4 lysate was prepared. The lysate was prepared 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). And placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

Transferring 50mg of the kiwi fruit root tissue sample into a 1mL glass homogenizer (Solarbio, YA 0850), adding 1mL of the lysate precooled on ice in the step (1), and smashing the crushed tissue for 20 times by using the glass homogenizer. The liquid in the homogenizer was transferred to a 2mL centrifuge tube (Axygen, MCT-200-C).

3) The cells were lysed.

4) Counting by microscopic examination.

The results are shown in Table 3.

Comparative example 24

This example follows the disruption of kiwi root tissue using a disruptor, KR5 lysate.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to a 2mL centrifuge tube (Axygen, MCT-200-C). 1mL of the lysate precooled on ice in step (1) was added. Rapid tissue cryodisruption homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Adding steel balls for crushing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) Counting by microscopic examination.

The results are shown in Table 3.

Comparative example 25

This example follows the disruption of kiwi root tissue using liquid nitrogen milling, KR5 lysate lysis.

2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2). Placing the prepared lysate on ice for precooling

2) And (4) crushing the sample.

Transferring 50mg of the kiwi fruit root tissue sample into a mortar, pouring a certain amount of liquid nitrogen, and grinding until the tissue is completely changed into powder. During this time, the addition of liquid nitrogen was noted to maintain the low temperature, and the milled powder was transferred to a 2mL centrifuge tube (Axygen, MCT-200-C) and 1mL of the ice-precooled lysate from step (1) was added.

3) The cells were lysed.

4) Counting by microscopic examination.

The results are shown in Table 3.

Comparative example 26

This example follows the disruption of kiwi root tissue using a disruptor, KR8 lysate.

1) KR8 lysate was prepared. The lysate was prepared according to the following ingredients:

2% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.1U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613). And (3) placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). 1mL of the lysate precooled on ice in step (1) was added. Mo Bai biological high throughput tissue grinder (onebo-48 p). Adding steel balls for breaking. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) Counting by microscopic examination.

5) And (4) RNA quality inspection.

Total RNA from the cell nucleus in step 3 was extracted using a plant Total RNA extraction kit (Tiangen Biochemical technology Ltd., DP 432). RNA was quality checked using an Agilent 2100 bioanalyzer.

The results are shown in Table 4

Comparative example 27

This example follows the disruption of kiwi root tissue using a disruptor, KR9 lysate.

1) KR9 lysate was prepared. The lysate was prepared 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 (murine), ACCURATE BIOLOGY, AG 11613). And placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). 1mL of the lysate precooled on ice in step (1) was added. Rapid tissue cryodisruption homogenizer (Shanghai Jingxin, JXFSTPRP-I-02). Adding steel balls for crushing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, the instrument was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) Counting by microscopic examination.

5) And (4) RNA quality inspection.

Total RNA from cell nuclei in step 3 was extracted using a plant Total RNA extraction kit (Tiangen Biochemical technology Ltd., DP 432). RNA was quality checked using an Agilent 2100 bioanalyzer.

The results are shown in Table 4

Comparative example 28

This example follows from the use of a disrupter to disrupt kiwi root tissue, KR10 lysate lysis fraction was 2% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.6U/mLRNase inhibitor (recombinant RNase inhibitor (murine source), ACCURATE BIOLOGY, AG 11613). The rest of the operation was the same as in comparative example 25.

The results are shown in Table 5

Comparative example 29

This example follows the use of a 70 μm (FALCON, 352350) screen to filter the nuclear suspension twice.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, the instrument was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (4) filtering.

The lysate was aspirated off serially using a pipette and filtered through a 70 μm sieve (FALCON, 352350). The filtrate was collected in a new 50mL centrifuge tube (Corning, 430828).

Note that the operation should be performed on ice.

5) And (5) filtering.

The filtrate obtained in step (4) was gradually aspirated by a pipette and filtered through a 70 μm filter (FALCON, 352350). The filtrate was collected into a new 15mL centrifuge tube.

Note that the operation should be performed on ice.

6) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) taking 5 mu L of the filtrate obtained in the step (5), uniformly mixing the filtrate with the DAPI dye solution 1:1, sucking 10ul of the mixture by using a pipette, adding the mixture into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by using a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 5

Comparative example 30

This example followed 1 filtration of the cell nucleus suspension using a 30 μm (Miltenyi Biotec, 130-110-915) screen, the rest of the procedure being identical to that of comparative example 29.

The results are shown in Table 5

Comparative example 31

This example followed the filtration of a nuclear suspension twice using a 30 μm (Miltenyi Biotec, 130-110-915) screen, and the rest of the procedure was the same as in comparative example 29.

The results are shown in Table 5

Comparative example 32

This example followed the filtration of nuclear suspensions using a 40 μm (FALCON, 352340) screen, the rest of the procedure being the same as in comparative example 29.

The results are shown in Table 5

Comparative example 33

This example followed the filtration of nuclear suspensions twice using a 40 μm (FALCON, 352340) screen, the rest of the procedure being the same as in comparative example 29.

The results are shown in Table 5

Comparative example 34

This example followed the removal of calcium oxalate crystals by filtration using a 70 μm (FALCON, 352350) screen.

1) And preparing a lysis solution. KR lysate was prepared according to the following ingredients:

2% (v/v) L-tartaric acid (Nanking Reagent, C0681014023), 15mM sodium citrate, 0.2% (m/v) saponin (Nanking Reagent, 8047-15-2), 0.2U/mL RNase inhibitor (recombinant RNase inhibitor (murine), ACCURATE BIOLOGY, AG 11613). And (3) placing the prepared lysate on ice for precooling.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). Adding 1mL of the lysate precooled on ice in the step (1), and adding steel balls for smashing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) And (5) filtering.

And (4) filtering all the filtrate in the step (5) by using a 70-micron (FALCON, 352350) screen, collecting the filtrate into a new 15-mL centrifuge tube, standing on ice for filtering until the liquid is completely filtered into the 15-mL centrifuge tube.

7) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (5) taking 5 mu L of the filtrate obtained in the step (6), uniformly mixing the filtrate with the DAPI dye solution 1:1, sucking 10ul of the mixture by a liquid transfer device, adding the mixture into a sample adding hole of a hemocytometer for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 6

Comparative example 35

This example was conducted in the same manner as in comparative example 32 except that calcium oxalate crystals were removed by filtration using a 40 μm (FALCON, 352340) screen.

The results are shown in Table 6

Comparative example 36

This example followed the removal of calcium oxalate crystals by filtration using a 30 μm (Miltenyi Biotec, 130-110-915) screen, the remaining procedure being identical to that of comparative example 32.

The results are shown in Table 6

Comparative example 37

This example follows the removal of calcium oxalate crystals using 100 Xg differential centrifugation.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) Differential centrifugation.

The filtrate obtained in step (5) was supplemented with precooled PBS to 10mL, and centrifuged at 100 Xg for 10min at 4 ℃. After centrifugation was complete, the supernatant was transferred to a new centrifuge tube and the pellet resuspended using 3mL of precooled PBS.

8) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) respectively taking 5 mu L of the supernatant and the heavy suspension obtained in the step (6), uniformly mixing the supernatant and the heavy suspension with the DAPI dye solution 1:1, sucking 10ul of the mixture by using a liquid transfer machine, adding the mixture into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by using a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 6

Comparative example 38

This example follows the removal of calcium oxalate crystals using density gradient centrifugation.

1) And (4) preparing a lysis solution. KR lysate was prepared according to the following ingredients:

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) Density gradient centrifugation.

Respectively preparing 50% (m/v) sucrose solution, 40% (m/v) sucrose solution, 20% (m/v) sucrose solution and 15% (m/v) sucrose solution.

Centrifuging the filtrate obtained in the step (5) at 4 ℃ for 10min at 500 Xg, discarding the supernatant after the centrifugation is finished, and resuspending the cell nuclear sediment by using 6mL of 15% (m/v) sucrose solution.

50% (m/v) sucrose solution, 40% (m/v) sucrose solution and 20% (m/v) sucrose solution were sequentially added from bottom to top in a Polyallemer centrifuge tube (Beckman, 355631), and then 6mL of cell nucleus solution was slowly added to the uppermost layer to form a density gradient.

18000rpm for 90min, and taking 50% (m/v) sucrose solution layer, 40% (m/v) sucrose solution layer and 20% (m/v) sucrose solution layer in three new centrifuge tubes.

7) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) respectively taking 5 mu L of the 50% (m/v) sucrose solution layer, the 40% (m/v) sucrose solution layer and the 20% (m/v) sucrose solution layer in the step (6), uniformly mixing with the DAPI dye solution 1:1, sucking 10ul of liquid by a liquid transfer machine, adding the mixture into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 6

Comparative example 39

This example follows the use of flow cytometry to remove calcium oxalate crystals.

2) And (4) crushing the sample.

50mg of kiwi root tissue samples were transferred to 2mL centrifuge tubes (Axygen, MCT-200-C). And (2) adding 1mL of the lysis solution precooled on ice in the step (1), and adding a steel ball for crushing. The centrifuge tube was placed on a crushing clamp and the wrench was tightened, and the rotation speed of the apparatus was adjusted to 1200rpm for 180 seconds. The instrument was started for tissue disruption.

3) The cells were lysed.

4) And (4) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) And (4) sorting by a flow cytometer.

And (5) adding a DAPI dye solution into the filtrate obtained in the step (5), wherein the final concentration of the DAPI is 5 mu g/mL. The sorting condition is set as that under the ultraviolet excitation light, a blue fluorescence signal (+) is collected.

7) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (4) respectively taking 5 mu L of the collected liquid in the step (6), uniformly mixing the collected liquid with the DAPI dye liquid 1:1, sucking 10ul of the mixed liquid by a liquid transfer device, adding the mixed liquid into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 6

Comparative example 40

This example follows the use of a MS sort column to remove calcium oxalate crystals.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

The tissue lysate from step (3) was transferred to a new 15mL centrifuge tube (Corning, 430790) using a pipette. 9mL of ice-cold PBS (Gibco, 10010-031) was added to the centrifuge tube. The mixture was allowed to stand on ice for 1 minute.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (4) filtering.

8) Counting by microscopic examination.

Calculating the cell nucleus concentration, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) respectively taking 5 mu L of the collected liquid in the step (6), uniformly mixing the collected liquid with the DAPI dye 1:1, sucking 10ul of the mixed liquid by a liquid transfer machine, adding the mixed liquid into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 6

Comparative example 41

This example followed the removal of calcium oxalate crystals using an LS sort column.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

The tissue lysate from step (3) was transferred by pipette into a new 15mL centrifuge tube (Corning, 430790). 9mL of ice-cold PBS (Gibco, 10010-031) were added to the centrifuge tube. The mixture was allowed to stand on ice for 1 minute.

Note that the operation should be performed on ice.

5) And (4) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

The 3mL of the resuspension from step (6) was added to LS Column (Miltenyi, 130-042-401) and the filtrate was collected in a new 15mL centrifuge tube and filtered by resting on ice until the liquid was completely filtered into the 15mL centrifuge tube.

9) Counting by microscopic examination.

The results are shown in Table 6

Comparative example 42

The washing conditions in this example were 100 Xg, 4 ℃,10 min washing.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (7) in a centrifuge, and centrifuging at 100 Xg for 10 minutes at 4 ℃.

9) And (6) quality inspection and microscopic examination counting.

The supernatant was aspirated and the concentration of RNA in the supernatant was measured using a NanoDrop microspectrophotometer and a fluorospectrophotometer.

Calculating the concentration of viable cell nuclei, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: taking 5 mu L of the heavy suspension liquid in the step (8), mixing the heavy suspension liquid with the DAPI dye solution 1:1 uniformly, sucking 10ul of the mixed solution by a liquid transfer machine, adding the mixed solution into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 7

Comparative example 43

The washing conditions in this example were 1000 Xg, 4 ℃,10 min washing.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (5) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (7) in a centrifuge, and centrifuging for 10 minutes at 1000 Xg at 4 ℃.

9) And (6) quality inspection and microscopic examination counting.

Calculating the concentration of viable cell nuclei, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: and (3) taking 5 mu L of the heavy suspension liquid obtained in the step (8), uniformly mixing the heavy suspension liquid with the DAPI dye solution 1:1, sucking 10ul of the mixture by a liquid transfer device, adding the mixture into a sample adding hole of a blood counting plate, counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 7.

Comparative example 44

The washing conditions of this example were 300 Xg, 4 ℃,10 min washing once.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (4) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (5) filtering.

8) The precipitate was collected by centrifugation.

9) And (6) quality inspection and microscopic examination counting.

The results are shown in Table 7

Comparative example 45

The washing conditions of this example were 300 Xg, 4 ℃,10 min washing three times.

2) And (4) crushing the sample.

3) The cells were lysed.

4) And (4) filtering.

Note that the operation should be performed on ice.

5) And (5) filtering.

Note that the operation should be performed on ice.

6) The precipitate was collected by centrifugation.

7) And (4) filtering.

8) The precipitate was collected by centrifugation.

After centrifugation was complete, the tube was carefully removed, the supernatant carefully decanted, and the pellet resuspended in 10mL ice-cold PBS.

9) The precipitate was collected by centrifugation.

After centrifugation was complete, the centrifuge tube was carefully removed, the supernatant carefully decanted, and the pellet resuspended using 10mL of ice-cold PBS.

10 ) the precipitate was collected by centrifugation.

Placing the centrifuge tube in the step (9) in a centrifuge, and centrifuging at 300 Xg for 10 minutes at 4 ℃.

11 Quality inspection, microscopic counting.

Calculating the concentration of viable cell nuclei, the agglomeration ratio and the impurity ratio by DAPI staining solution and microscopic examination: taking 5 mu L of the heavy suspension liquid in the step (10), mixing the heavy suspension liquid with the DAPI dye solution 1:1 uniformly, sucking 10ul of the mixed solution by a liquid transfer machine, adding the mixed solution into a sample adding hole of a blood counting plate for counting, and observing the number of cell nuclei in a counting area by a microscope. Fluorescence is shown as nuclei.

The results are shown in Table 7

Comparative example 46

The experimental sample of this example was 50mg of Arabidopsis thaliana leaves, and the specific operation was the same as that of example 1.

The results are shown in Table 8

Comparative example 47

The experimental sample of this example is 50mg of Arabidopsis root, and the specific operation is the same as that of example 1.

The results are shown in Table 8

Comparative example 48

The experimental sample of this example is 50mg of corn embryo, and the specific operation is the same as that of example 1.

The results are shown in Table 8

Table 1 results of examples 1 to 4

The invention discloses a method suitable for sequencing single cell nucleus of a kiwi fruit root tissue, which comprises a method for extracting the cell nucleus of the kiwi fruit root tissue and a method for optimizing cell nucleus suspension. The technical method provided by the invention can introduce the single cell sequencing technology into related researches on kiwi fruit molecular mechanism and breeding, and can effectively and efficiently promote the research progress.

The invention relates to a preparation method of a single cell nucleus suitable for kiwi root tissues, which mainly comprises a lysate, wherein the components of the lysate are 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 0.2-0.4U/mL RNase inhibitor (mouse source), ACCURATE BIOLOGY, AG 11613). Extracting cell nucleus from tissue by crushing sample and cracking cell, and performing suspension optimization treatment such as screen filtering and centrifugal washing to obtain the product with the number of more than 1 × 10 ⁶ The cell nucleus suspension with fragmentation rate less than 10% and agglomeration rate less than 4% meets the quality requirement of 10x single cell sequencing on the cell nucleus suspension. The purpose of using a high-throughput single-cell sequencing technology to research the kiwi root tissue is achieved.

In order to obtain the method suitable for sequencing the single cell nucleus of the kiwi fruit root tissue, a series of explorations in the aspects of lysate formula, experimental operation steps, optimization method and the like are carried out, and the content of the kiwi fruit root tissue is finally obtained by consulting data and verifying. Firstly, through comparison examples 1-3, the preparation of cell nucleus suspensions of arabidopsis thaliana leaves, kiwi fruit leaves and kiwi fruit root tissues is respectively carried out by using a method in Identification of open chromosome Regions in Plant Genomes UsingATAC-Seq, and the result shows that the method in the document can only complete the preparation of the cell nucleus suspensions of the arabidopsis thaliana leaf tissues and cannot complete the preparation of the cell nucleus suspensions of the kiwi fruit leaves and kiwi fruit root tissues; and the prepared arabidopsis thaliana leaf cell nucleus suspension has a slightly high impurity ratio, and although the minimum requirement of single cell sequencing on a sample is met, the risk exists when the single cell sequencing is carried out. In comparative examples 4 to 6, the method of Isolation of Plant Root Nuclei for Single Cell RNA Sequencing was used to perform a Cell nucleus suspension preparation experiment of arabidopsis Root tissue, kiwi leaf tissue and kiwi Root tissue by using the existing commercial kit, and the results show that the method in the literature can only complete the preparation of Cell nucleus suspension of arabidopsis Root tissue and cannot complete the preparation of Cell nucleus suspension of kiwi leaf tissue, and the results show that the number of kiwi Root Nuclei extracted by the existing commercial kit is very small, and the kiwi Root tissue Cell nucleus suspension satisfying Single Cell Sequencing cannot be successfully prepared. Both methods show that the prepared cell nucleus has a small number and the prepared cell nucleus has a great amount of impurities. The key to guarantee the number of extracted cell nuclei is that the lysis solution fully lyses tissues and simultaneously a proper buffer solution guarantees the integrity of the cell nuclei.

Firstly, through comparison examples 7-20, a buffer system suitable for a kiwi root tissue is explored, and the result is shown in table 1, and the buffer system in the existing scheme cannot be suitable for a kiwi root tissue cell nucleus extraction experiment; the kiwi fruit grows in the wild, the cell growth mechanism is greatly different from that of a model organism, and calcium oxalate, aldehydes and the like can be enriched in kiwi fruit root cells. Compared with buffer components needing stronger buffer capacity such as model organism Arabidopsis thaliana and the like, tris-HCl, MOPS and the like are not suitable for common buffer components. Increasing the concentration promotes the reaction of Tris with aldehydes and various enzymes in solution, resulting in an unstable lysis system. Finally, a buffer system constructed by L-tartaric acid and sodium citrate is selected through exploration. L-tartaric acid is commonly used as an acidulant for beverages and other food products, has strong buffering capacity and plays a major buffering role in the present invention; meanwhile, tartaric acid has stronger reducibility, and plays a role in protecting nucleic acid in cell nucleus in the 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 cell nucleus is broken due to the change of the pH of the buffer; when the concentration of the buffer component is high, the cell nucleus is agglomerated due to the high concentration of ions in the buffer, and the experiment fails. Therefore, the cell nucleus preparation buffer component in the invention is 2-3% (v/v) L-tartaric acid (Nanjing Reagent, C0681014023) and 15-25 mM sodium citrate.

The cell membrane is damaged when the plant cell nucleus is extracted, the basic structure of the cell membrane is phospholipid bilayer, and the aim of releasing the cell nucleus can be achieved by using a detergent to damage the phospholipid bilayer. In comparative examples 7 to 10, the formulation of the lysate was explored, and the experiment was performed using lysis components of different properties and different working principles, and the results showed that a large number of nuclei were broken and sufficient and complete nuclei could not be collected when SDS (sodium dodecyl sulfate) and a strong sodium deoxycholate detergent were used as the lysis components; when the digitonin weak detergent is used as a cracking component, the digitonin cannot dissolve membrane proteins, the structure of a cell membrane cannot be fully damaged, and relatively more cell nuclei can be collected but still the requirement of single cell sequencing on the number of the cell nuclei cannot be met. Triton X-100 belongs to a mild detergent, is derived from polyoxyethylene, contains an alkylphenyl hydrophobic group, has the capacity of combining lipid molecules between sodium dodecyl sulfate and digitonin, can be used as a cosolvent of membrane protein, and has more cell nuclei obtained by cracking than digitonin, but Triton X-100 can cause damage to a nuclear membrane when acting on a cell membrane, and sufficient cell nuclei cannot be obtained for a single-cell nuclear sequencing experiment. The method for preparing the cell nucleus by dissolving the cell membrane by using the common detergent is not suitable for the cell nucleus preparation experiment of the kiwi root tissue, and the saponin is commonly used as a medical raw material and is mainly used for synthesizing hormone medicaments. The saponin is composed of saponin and sugar, uronic acid or other organic acid, and the saponin has the function of emulsifying lipid molecules. The results of the comparative examples show that when saponin is used as a cracking component, the yield of cell nuclei is remarkably improved compared with SDS, triton X-100 and the like; meanwhile, saponin does not cause protein denaturation, so that saponin serving as a cracking component does not damage nuclear membranes of nuclei. The results of the comparative examples show that the number of nuclei is significantly reduced when the concentration of saponin is less than 2%, because saponin itself contains sugar in organic acid, etc., and when the concentration of saponin is more than 4%, the stability of the lysis system is affected and the number of nuclei is reduced. Therefore, the concentration range of the saponin in the lysis solution is 2-4% (m/v).

TABLE 2 comparative examples 1 to 20

The plant cell membrane is also coated with cell walls, the cell walls are formed by cellulose, hemicellulose, pectin and other components, the kiwi fruit plant is a large-scale deciduous vine, the supporting strength of the cell walls is far higher than that of arabidopsis thaliana, and therefore stronger crushing conditions are needed to help obtain more cell nucleuses in the method for crushing the cell walls. Next, by comparing the effects of different treatment methods including blade chopping, scissors chopping, homogenization, disrupter disruption, mortar grinding, etc. on the release of cell nuclei, it was shown by comparing the results of examples 21 to 25 that the experimental methods of blade chopping, scissors chopping, mortar grinding, etc. all gave a lower number of cell nuclei than the homogenization method, mortar liquid nitrogen grinding is the most sufficient method for tissue disruption, but mortar grinding also caused cell nuclei to be disrupted while breaking cell walls. The results of the comparative examples are analyzed, and the crushing mode of the crusher assists the lysate to fully release the cell nucleus and simultaneously has less damage to the cell nucleus, so that the method is the best method for extracting the cell nucleus of the kiwi root tissue.

TABLE 3 comparative examples 21 to 25

When a plant cell nucleus is subjected to a single cell transcriptome experiment, the integrity of mRNA in a cell needs to be ensured, and after the cell is crushed, RNA enzyme in cytoplasm is released into a buffer system to degrade the mRNA, so that the mRNA of the cell nucleus needs to be protected by an RNA enzyme inhibitor and a reducing agent, and the content difference of the RNA enzyme in different species and tissues is large, the results of comparative examples 26 to 28 show that 0.2 to 0.4U/mL of RNase inhibitor (recombinant RNase inhibitor (murine source), ACCURATE BIOGY and AG 11613) can well protect the integrity of the mRNA aiming at 50 to 100mg of kiwi root tissues, the obtained result of the RNase inhibitor has no obvious difference, the cost is increased, and the waste of reagents is also caused, so the concentration of the RNA enzyme inhibitor in KR lysate is 0.2 to 0.4U/mL.

TABLE 4 comparative examples 26 to 28

The crude cell nucleus suspension obtained by extracting the tissue after the treatment of the lysate contains a large amount of impurities such as cell fragments and mRNA, organelle and the like released by cell disruption. The large fragments of the cell nucleus suspension under the condition can not be subjected to a single cell sequencing experiment, the requirement of single cell sequencing can be met only after the large fragments are removed, larger tissues and cell fragments are firstly removed by filtration, screens with different apertures and different filtration times are verified through comparative examples 29-33, the yield and the impurity removal efficiency of the crude cell nucleus suspension are filtered, the result of the comparative example 29 shows that the 70 mu m screen can not well achieve the purpose of impurity removal, the impurity reduction of the suspension filtered by the 70 mu m screen is not obvious, and the two times of filtration still have no obvious effect. The fragments of the sample crushed by the crusher larger than 70 μm are less, in comparative examples 30-31, the effect of filtering the suspension of the kiwifruit root crude cell nuclei by using the 30 μm pore size screen is verified, and the result shows that the 30 μm cell screen can cause a great amount of loss of the cell nuclei, the filtering process is affected by high fragment rate, the filtering time is long, the risk of RNA degradation in the cell nuclei is increased, and therefore, the 30 μm screen cannot be used as a method for purifying the suspension of the kiwifruit root crude cell nuclei. The results of the comparative examples 32 to 33 show that the 40 μm cell screen can rapidly remove impurities from the crude cell nucleus suspension, and the best method for purifying the crude cell nucleus suspension of the kiwi fruit root is to filter the crude cell nucleus suspension by the 40 μm cell screen twice, the impurity removal efficiency is as high as 80%, and the cell nucleus yield is higher than 70%, so that the selection not only rapidly and effectively reduces the fragment rate, but also has higher cell nucleus yield.

TABLE 5 COMPARATIVE EXAMPLES 29 to 33

During the growth process of kiwi plants, a large amount of calcium oxalate needle crystals are enriched in cells/tissues, and the calcium oxalate needle crystals are an important component part for plant defense and self-protection. The calcium oxalate crystals are released into the cell nucleus suspension after the tissue is broken (fig. 3 shows that needle-shaped calcium oxalate crystals in the cell nucleus suspension), needle-shaped crystals exist in the cell suspension after the cell nucleus suspension is filtered by the screen, and the impurities cannot be effectively filtered in the screen filtration process no matter the particle size is 30 mu m or 70 mu m. The existence of calcium oxalate crystals can lead to the incapability of carrying out single cell sequencing, the calcium oxalate crystals exist in the tissues of the roots of kiwi fruits as needle-shaped crystals with different thicknesses, and the length of the calcium oxalate crystals is more than 70 mu m according to microscope observation. The effect of removing calcium oxalate crystals by the 70 μm cell sieve was confirmed by comparative example 34, and the results showed that the 70 μm cell sieve could not effectively remove calcium oxalate needle crystals. The effect of removing calcium oxalate crystals by a 40 μm, 30 μm sieve was verified by comparative examples 35 to 36, and the results showed that calcium oxalate crystals could not be removed by decreasing the mesh size. The calcium oxalate needle crystals easily pass through the screen with the liquid flow, and therefore the screen filtration method cannot remove the calcium oxalate needle crystals. Next, through comparative examples 37 to 39, the effects of removing calcium oxalate crystals by using a differential centrifugation method, a density gradient centrifugation method and a flow cytometry method are respectively verified, and the results show that the differential centrifugation method cannot achieve the effect of removing the calcium oxalate crystals and can cause a great amount of nuclear loss; the density gradient centrifugation can cause a great amount of loss of cell nuclei, the continuous sequencing of single cell nuclei has risks, the density gradient centrifugation cannot purify the cell nuclei well due to the fact that the specific gravity of impurities is similar to that of the cell nuclei, and the differential centrifugation method and the density gradient centrifugation method cannot achieve the purpose of purifying the cell nuclei. The existence of calcium oxalate needle crystals can directly cause the blockage of a flow cytometer, and sufficient cell nucleuses cannot be collected for experiments. Comparative examples 40 to 41 the search for optimizing the cell nucleus suspension was performed using MACS sorting columns, which are generally used for sorting specific cell types of animals by binding specific antigens on the surface of target cell membranes with antibodies having magnetic beads under magnetic field, in the present invention, small sized cell nuclei can pass through the sorting columns along with liquid flow, and needle shaped calcium oxalate crystals are left in the sorting columns and cannot pass through along with liquid flow, by using the needle shape morphology of calcium oxalate crystals and the stacking characteristics of nanomaterials in the sorting columns under non-magnetic field, so that the purpose of purifying cell nuclei can be achieved, the cell nucleus yield can be ensured, and the problem of optimizing the single cell nucleus suspension can be solved. The results show that all indexes of the filtrate obtained by filtering the mononuclear cell suspension through the MS sorting column all meet the requirements of a 10 XGenmics platform on single cell sequencing.

TABLE 6 comparative examples 34 to 41

After the removal of calcium oxalate crystals is completed, the data quality needs to be improved by removing free mRNA in the cell nucleus suspension through washing, and meanwhile, the concentration of the cell nucleus suspension needs to be adjusted, and the washing conditions are verified in comparative examples 42 to 45. The results show that too little centrifugal force can cause great loss of cell nucleus, too much centrifugal force can cause damage of cell nucleus, and too many washing times can cause breakage of cell nucleus; the washing condition is that at 4 ℃, centrifugation is carried out for 10 minutes at 300 Xg-500 Xg, washing is carried out for 2 times, free mRNA in the cell nucleus suspension is basically removed, and meanwhile, the cell nucleus yield is higher than that of other methods, so that the method is suitable for washing the cell nucleus suspension of the kiwi fruit root tissue.

TABLE 7 COMPARATIVE EXAMPLES 42 to 45

The cell walls of plants of different species and tissues have different structures, and the structures of cell contents are also different, and comparative examples 46 to 48 verify the results of preparing arabidopsis thaliana leaves, arabidopsis thaliana root tips and maize embryo cell nucleus suspensions by using KR lysate and the cell nucleus extraction and optimization method in the patent. The results show that the cell nucleus preparation method, the corresponding lysate formula and the cell nucleus optimization method in the patent correspond to the kiwi root tissue one by one, the preparation of the cell nucleus suspension of the kiwi root tissue can be completed only by carrying out the steps in sequence, and the method is not suitable for other species and tissues.

TABLE 8 comparative examples 46 to 48

In conclusion, the KR lysate obtained in comparative examples 1-28 comprises 2-3% (v/v) L-tartaric acid (C0681014023), 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin (Nanjing Reagent, 8047-15-2), 0.2-0.4U/mL RNase inhibitor (murine), ACCURATE BIOLO, and the manner of assisting the release of the kiwi root nuclei is that the disruption instrument disrupts, the experimental method for optimizing the nuclei is obtained in comparative examples 29-41, wherein a 40 μm screen is used for filtering twice to remove large fragments, an MS sorting column is used for removing calcium oxalate, the comparative examples 42-45 verify that a suspension of the nuclei is high in yield, the KR-environment-removed kiwi root tissue nuclei is used for washing, the kiwi root tissue nuclei is used for washing 300-500 times, the nuclear suspension is used for washing twice, and the nuclear suspension is used for preparing the kiwi root tissue nuclei, and the suspension is used for washing the nuclei twice, and the comparative examples 46-48-10-g nuclear suspension is used for preparing the kiwi nucleus suspension.

The preparation method of the kiwi root tissue cell nucleus suitable for single cell sequencing, which is described by the invention, can effectively prepare the cell nucleus suspension required for carrying out a 10x Genomics platform single cell transcriptome sequencing experiment. According to the preparation method of the kiwi fruit cell nucleus suspension suitable for single cell transcriptome sequencing, the kiwi fruit root, KR lysate, cell nucleus suspension filtering operation, cell nucleus suspension washing operation and single cell transcriptome sequencing result are in one-to-one correspondence, namely, the kiwi fruit root, KR lysate, cell nucleus suspension filtering operation, cell nucleus suspension washing operation and single cell transcriptome sequencing result are in compatibility. For example, the kiwi root, KR lysate, and cell nucleus suspension filtration operations mentioned in embodiments 1 to 4 of the present invention should be used according to the method indicated in the present invention, so that the cell nucleus suspension can be effectively prepared, and each index of the prepared cell nucleus suspension can meet the single cell transcriptome sequencing requirements. The preparation method of the kiwi root cell nucleus suspension suitable for single cell transcriptome sequencing is scientific and strict in operation and high in repeatability, can effectively and efficiently separate cell nucleus suspensions with sufficient quantity, low impurity rate and extremely low environmental RNA from kiwi root tissues, and the obtained cell nucleus suspensions can reach various indexes of single cell transcriptome sequencing of a 10x Genomics platform.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the present invention without departing from the spirit and scope of the inventive concept and are intended to be protected by the following claims.

Claims

1. A preparation method of kiwi root tissue cell nucleuses suitable for single cell sequencing is characterized by comprising the following specific steps:

1) Preparing KR lysate: firstly, preparing KR lysate, and precooling the prepared solution;

2) Crushing a sample: transferring a fresh or frozen kiwi fruit root tissue sample into a centrifuge tube, adding the pre-cooled KR lysate and steel balls, and placing the sample on a crusher according to an instrument operation instruction to crush the tissue;

3) Cell lysis: after crushing, putting the centrifuge tube on ice for incubation and cracking;

4) Filtering with a screen: transferring the cracked tissue lysate into a centrifuge tube, adding precooled PBS, standing on ice for 1 minute, sucking out the tissue lysate, adding the tissue lysate into a filter screen, and filtering to obtain a filtrate;

5) Filtering with a screen: filtering the filtrate obtained in the step 4) again by using a filter screen;

6) Centrifuging and collecting precipitates: centrifuging the filtrate obtained in the step 5), discarding the supernatant, and resuspending the precipitate by using PBS;

7) And (3) filtering: adding all the precipitate resuspension in the step 6) into a separation column for filtering;

8) And (3) centrifuging and collecting precipitates: the filtrate from step 7) was centrifuged, the supernatant discarded and the pellet resuspended in PBS.

2. The method according to claim 1, wherein in step (1), the KR cleavage solution comprises the following components (final concentrations): 2-3% (v/v) L-tartaric acid, 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin and 0.2-0.4U/mL recombinant RNase inhibitor.

3. The method according to claim 1, wherein in the step (2), the disruptor is a myrica esculenta biological high-throughput tissue disruptor; the crushing conditions are 1200-1300rpm and 170-180 s; and adding 50-100 mg of kiwi root tissues into 1ml of KR lysate.

4. The method according to claim 1, wherein the incubation period in the step (3) is 7 to 8 minutes.

5. The method according to claim 1, wherein in the step (4), the screen is FALCON 40 μm Cell Strainer with a pore size of 40 μm; the whole filtering process is operated at 0-5 ℃.

6. The method according to claim 1, wherein in the step (6), the temperature of the centrifugation is 4 to 6 ℃, the time of the centrifugation is 10 to 15 minutes, and the centrifugal force is 300 to 500 Xg.

7. The method of claim 1, wherein in step (7), the sorting Column is a Miltenyi MS Column; the filtration is carried out at 0-5 ℃ in the whole process.

8. The method according to claim 1, wherein in the step (8), the centrifugation temperature is 4 to 6 ℃, the centrifugation time is 10 to 15 minutes, and the centrifugal force is 300 to 500 Xg.

9. KR lysate, characterized in that KR lysate contains the following components (final concentration): 2-3% (v/v) L-tartaric acid, 15-25 mM sodium citrate, 0.2-0.4% (m/v) saponin and 0.2-0.4U/mL recombinant RNase inhibitor.

10. A reagent/kit comprising KR lysate according to claim 9.

11. Use of KR lysate according to claim 9 or reagent/kit according to claim 10 in the preparation of a method for actinidia root tissue nuclei suitable for single cell sequencing, actinidia root tissue nuclei transcriptome sequencing, actinidia root tissue nuclei ATAC sequencing studies.