CN115505629A - Method for preparing single cell nucleus of frozen tissue - Google Patents
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Abstract
The invention discloses a method for preparing single cell nuclei of frozen tissues, which comprises the steps of directly cracking the nuclei in the frozen tissues, then marking and capturing high-quality nuclei, constructing a library by a labeled nuclear transcriptome, and realizing single cell research in cold tissues through second-generation sequencing and data analysis. The single cell research scheme suitable for the frozen tissue solves the problem that the traditional single cell experiment method cannot carry out the single cell experiment of the frozen sample. The method has better technical performance, and the indexes of high-quality cell number, cell gene median, total detected gene number and the like in the analysis result of the method are better and can be comparable with the experimental result of a fresh tissue.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing a single cell nucleus of a frozen tissue.
Background
Single cell sequencing (scRNA-seq) has become an important tool for exploring cell types, molecular changes and cell functions in complex tissues. However, the single cell experiment requires the preparation of single cell suspension from fresh tissue to obtain higher cell activity and cell quality, but the frozen tissue sample at-80 ℃ cannot be applied to single cell sequencing, because the frozen tissue sample at-80 ℃ cannot prepare high quality cell suspension, which is a major obstacle limiting the wide application of single cell technology. Therefore, it is necessary to establish a single cell experimental method for frozen tissues to solve the problem of single cell application of frozen tissue samples.
However, at present, no mature method is available for carrying out dissociation experiments and single cell capture experiments of frozen tissues, conventional single cell dissociation experiments cannot obtain high-activity cell suspensions from frozen tissues, and even if single cell marking experiments are performed on the low-activity cell suspensions, indexes such as high-quality cell number, cell gene median, total detected gene number and the like in subsequent analysis results are poor, and the indexes cannot be comparable with the experimental results of fresh tissues.
Disclosure of Invention
Aiming at the technical problems existing at present, the invention aims to provide a method for preparing single cell nucleuses of frozen tissues, which solves the problem of single cell application of frozen samples. The invention develops the nuclear transcriptome sequencing (snRNA-seq) on the single cell level, directly cracks cell nuclei in frozen tissues, then marks and captures the high-quality cell nuclei, constructs a library for the nuclear transcriptome with a label, and realizes the single cell research in the cold tissues through second-generation sequencing and data analysis. The method is suitable for a single cell research solution of frozen tissues and has better technical performance.
In order to achieve the purpose, the invention adopts the following technical means:
a method for preparing single cell nuclei of frozen tissue, comprising the steps of:
s1, adding frozen tissues into a first buffer solution for cleaning, shearing, settling and cleaning for 2 times, and removing supernatant to obtain a first precipitate;
s2, adding a precooled lysate into the first precipitate, pre-lysing on ice, and then homogenizing by using a homogenizer to obtain homogenate; wherein the lysate is precooled at 0-4 ℃, and the amount of the lysate is 1-3mL;
s3, transferring the homogenate to ice for incubation, adding a precooled bovine serum albumin solution (BSA solution), blowing and beating for 6-8 times by using a Pasteur pipette, and stopping the reaction to obtain a first mixture; wherein the incubation time is 4-8min; the adding amount of the bovine serum albumin solution is 1-3mL, and the mass fraction is 4%;
s4, centrifuging the first mixture at the temperature of 4 ℃ for 5min by 300g, and removing a supernatant to obtain a second precipitate;
s5, resuspending the second precipitate by using a lysate and a bovine serum albumin solution, and incubating for 2-4min on ice to obtain a second mixture; wherein, the lysis solution: bovine serum albumin solution volume ratio = 125;
s6, removing debris in the second mixture by adopting a flow sorting instrument (FACS) to obtain a third precipitate; wherein the debris comprises cellular debris, free nucleic acids, non-cellular contaminants, and the like.
S7, adding lysis solution and BSA solution into the third precipitate for resuspension, incubating on ice for 5min, centrifuging at the temperature of 4 ℃ for 5min at 300g, and removing supernatant to obtain cell nucleus precipitate; wherein the addition amount of the lysis solution is 4mL; wherein, the lysis solution: bovine serum albumin solution volume ratio =125
S8, adding a second buffer solution for resuspending the cell nucleus sediment into the cell nucleus sediment for resuspension, sieving the cell nucleus sediment by a 20-micron cell sieve, and collecting an effluent cell suspension, namely the single cell nucleus of the frozen tissue;
further, the specific process of step S6 is as follows:
s61, precooling a sample chamber and a sorting and collecting device of a cell sorter to 4 ℃;
s62, filtering 200 mu l of the second mixture to obtain filtered cell nuclei, transferring the cell nuclei into a flow tube, and taking a filter cap of the flow tube as a non-staining control, namely a baseline for cell nucleus sorting;
s63, dyeing the remaining second mixture with propidium iodide, and then placing on ice for 10min; wherein the final concentration of propidium iodide is 10 μ g/ml;
s64, sorting the second mixture, wherein the sorter uses a 70nm nozzle with a default pressure of 20psi; exciting propidium iodide with a 488nm laser, and detecting by using a 600nm high-pass filter and a 610/20nm band-pass filter; collecting the sorted cell nuclei, placing the cell nuclei in a 15ml conical tube, adding 1ml PBS, and placing the cell nuclei on ice;
s65, centrifuging at the rotation speed of 1000g for 10min at 4 ℃ after sorting is finished; removing supernatant, adding 3ml PBS, and resuspending cell nucleus, wherein the resuspension method is a gentle spin method; then centrifuging at the rotation speed of 1000g for 10min at 4 ℃; after removing the supernatant, 30-100. Mu.l of PBS was added to further resuspend the nuclei, thereby obtaining a first nuclear pellet. To prevent nuclear membrane leakage, the cell nuclei were resuspended using a gentle spin method. Under a fluorescent microscope, propidium iodide or DAPI staining can easily detect damaged nuclei, but discrete leakage cannot.
Further, the method for preparing single-cell nuclei of frozen tissue further comprises the step of performing RNA quality detection on the frozen tissue; wherein the quality detection instrument is an Agilent 2100 biomass detection instrument, the detection standard is that the RNA integrity value is more than or equal to 6 (RIN is more than or equal to 6), and the frozen tissues which are unqualified to be detected are re-sampled.
Further, the method for preparing the single cell nucleus of the frozen tissue further comprises the step of performing data analysis by using cellrange and saurat software.
Further, the method for preparing the single cell nucleus of the frozen tissue further comprises a process of measuring and counting the concentration of the cell suspension by using a blood cell counting plate or a Countess II automatic cell counter for the prepared single cell nucleus, wherein the optimal concentration is adjusted to 700-1200nuclei/ul;
preferably, in step S1, the buffer is PBS buffer;
preferably, the lysate is prepared by mixing EZ lysate and PBS; wherein the volume ratio of the EZ lysate to the PBS is 1:1; EZ lysate Cat number NUC-101, purchased from Sigma-Aldrich; the homogenizer was a Dounce homogenizer, purchased from Kimble Chase, and numbered 885302-0002;
preferably, the mass concentration of the BSA solution is 4%;
preferably, the second buffer consists of 1 XPBS, 0.07wt% BSA and 0.1wt% RNase inhibitor; wherein the RNase inhibitor is RNase inhibitor, cat # AM2696, available from Life Technologies.
Preferably, the frozen tissue is a tissue sample of a frozen animal species.
The invention also provides an application of the single cell nucleus prepared by the method for preparing the frozen tissue single cell nucleus, which comprises the following steps:
s11, capturing qualified cell nucleus suspension through a 10x Genomics single cell platform, marking transcripts in the cell nucleus, carrying out reverse transcription, and constructing a library; wherein, the library is constructed according to 10x Genomics marking and library establishing experimental procedures;
s12, after the library is constructed, firstly using the qubit3.0 to carry out preliminary quantification, diluting the library to 1ng/ul, then using Agilent 2100 to detect the insert size of the library, after the insert size meets the expectation, using a Bio-RAD CFX 96 fluorescence quantitative PCR instrument and a Bio-RAD KIT iQ SYBRGRN to carry out Q-PCR, and accurately quantifying the effective concentration of the library to ensure the quality of the library. Quality-qualified library Illumina platform (Illumina Novaseq) TM 6000 ) for sequencing. Wherein the effective concentration of the library is more than 10nM, and the sequencing strategy is PE150.
A method of the present invention for preparing single cell nuclei of frozen tissue is also applicable to fresh tissue, large cell-containing tissue, or hard-to-dissociate tissue (e.g., fat, cartilage, etc.).
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the single cell research scheme suitable for the frozen tissue solves the problem that the traditional single cell experiment method cannot carry out the single cell experiment of the frozen sample. The method has better technical performance, and the indexes such as high-quality cell number, cell gene median, total detected gene number and the like in the analysis result of the method are better and can be comparable with the experimental result of a fresh tissue. The method is convenient and effective, and is suitable for popularization and application.
Drawings
FIG. 1 shows Propidium Iodide (PI) staining patterns of the nuclear suspension of example 2 of the present invention;
FIG. 2 shows the quality test results of the snRNA-seq data analysis of frozen mouse cortex in example 2 of the present invention;
FIG. 3 shows the result of cell identification by analyzing snRNA-seq data of frozen mouse cortex according to example 2 of the present invention;
FIG. 4 shows a cell/nucleus number map of scRNA-seq and snRNA-seq detection of example 3 of the present invention;
FIG. 5 is a graph showing the average number of sequenced fragments per cell for scRNA-seq and snRNA-seq of example 3 of the present invention;
FIG. 6 shows the cellular gene median plots for the detection of scRNA-seq and snRNA-seq of example 3 of the present invention;
FIG. 7 shows a graph of the total gene counts for scRNA-seq and snRNA-seq detection of example 3 of the present invention;
FIG. 8 shows a single cell nuclear transcriptome flow chart.
Detailed Description
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety and their equivalent family patents are also incorporated by reference, especially as they disclose definitions relating to synthetic techniques, products and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing numerical values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), 1 unit is considered to be 0.0001,0.001,0.01 or 0.1, as appropriate. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
When used with respect to chemical compounds, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively) unless expressly specified otherwise. In addition, unless explicitly stated otherwise, the use of the terms "a", "an" or "the" are intended to include the plural forms as well.
The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of … …" excludes any other components, steps or processes from the scope of any such term as are hereinafter recited, out of those necessary for performance. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and materials cited therein are hereby incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Example 1
A method for preparing single cell nuclei of frozen tissue, comprising the steps of:
s1, adding frozen tissues into a buffer solution, shearing, settling and cleaning for 2 times, and removing a supernatant to obtain a first precipitate;
s2, adding a precooled lysate into the first precipitate, pre-lysing the first precipitate on ice, homogenizing the first precipitate by using a homogenizer, and gently blowing and beating the first precipitate upwards and downwards for 5-6 times by using a liquid transfer device to obtain homogenate; wherein, the lysate is precooled at 4 ℃, and the consumption of the lysate is 2mL;
s3, transferring the homogenate to ice for incubation, adding a precooled bovine serum albumin solution (BSA solution), blowing and beating for 7 times by using a Pasteur pipette, and stopping the reaction to obtain a first mixture; wherein the incubation time is 6min; the adding amount of the bovine serum albumin solution is 2mL, and the mass fraction is 4%;
s4, centrifuging the first mixture at the temperature of 4 ℃ for 5min by 300g, and removing a supernatant to obtain a second precipitate;
s5, resuspending the second precipitate by using a lysate and a bovine serum albumin solution, and incubating for 3min on ice to obtain a second mixture; wherein, the lysis solution: bovine serum albumin solution volume ratio =125
S6, removing debris in the second mixture by adopting a flow sorting instrument (FACS) to obtain a third precipitate; wherein the FACS is a BD FACSAria type II cell sorter. The method comprises the following specific steps:
s01, precooling a sample chamber and a sorting and collecting device of a BD FACSAria II type cell sorter to 4 ℃;
s02, filtering 200 mu l of the second mixture to obtain filtered cell nuclei, transferring the cell nuclei into a flow tube, and taking a filter cap of the flow tube as a non-staining control, namely a baseline for cell nucleus sorting;
s03, dyeing the remaining second mixture with propidium iodide, and then placing on ice for 10min; wherein the final concentration of propidium iodide is 10 μ g/ml;
s04, sorting the second mixture, wherein the sorter uses a 70nm nozzle with a default pressure of 20psi; exciting propidium iodide with a 488nm laser, and detecting by using a 600nm high-pass filter and a 610/20nm band-pass filter; collecting the sorted cell nuclei, placing the cell nuclei in a 15ml conical tube, adding 1ml PBS, and placing the cell nuclei on ice;
s05, centrifuging at the rotation speed of 1000g for 10min at 4 ℃ after sorting is finished; removing supernatant, adding 3ml PBS, and resuspending cell nucleus, wherein the resuspension method is a gentle spin method; then centrifuging the mixture for 10min at the rotating speed of 1000g at the temperature of 4 ℃; after removing the supernatant, 30-100. Mu.l of PBS was added to further resuspend the nuclei, resulting in a third pellet.
S7, adding lysis solution and BSA solution into the third precipitate for resuspension, incubating on ice for 5min, centrifuging at the temperature of 4 ℃ for 5min at 300g, and removing supernatant to obtain cell nucleus precipitate; wherein the addition amount of the lysis solution is 4mL; lysis solution: bovine serum albumin solution volume ratio =125
S8, resuspending the cell nucleus sediment by using a second buffer solution, screening by using a 20um cell screen, and collecting an effluent cell suspension, namely the cell nucleus of the frozen tissue;
further, the concentration of the cell suspension is measured and counted by adopting a blood cell counting plate or a Countess II automatic cell counting instrument, and the optimal concentration is adjusted to 700-1200 nucleolei/ul; single cell sequencing was performed on a qualified nuclear suspension as follows:
s11, capturing qualified cell nucleus suspension through a 10x Genomics single cell platform, marking transcripts in the cell nucleus, carrying out reverse transcription, and constructing a library; wherein, the library is constructed according to 10x Genomics marking and library establishing experimental procedures;
s12, after the library is constructed, firstly, using Qubit3.0 to carry out preliminary quantification, diluting the library to 1ng/ul, then using Agilent 2100 to detect the insert size of the library, after the insert size meets the expectation, using a Bio-RAD CFX 96 fluorescence quantitative PCR instrument and a Bio-RAD KIT iQ SYBRGRN to carry out Q-PCR, and accurately quantifying the effective concentration of the library to ensure the quality of the library. Quality-qualified library Illumina platform (Illumina Novaseq) TM 6000 ) for sequencing. Wherein the effective concentration of the library is more than 10nM, and the sequencing strategy is PE150.
Example 2
This example provides a single cell nuclear assay of frozen mouse brain tissue.
Taking a C57BL/6 mouse of 7 weeks old, performing a brain perfusion experiment, then performing an operation to obtain a fresh cerebral cortex, cleaning the cerebral cortex with PBS, then quickly freezing the frozen tissue by adopting liquid nitrogen to obtain a frozen tissue, firstly taking 5mg of the tissue to extract RNA, performing RNA quality detection by using an Agilent 2100 biomass detector, wherein the RNA integrity value (RIN) =8.8, the quality is qualified, and then performing a single cell nucleus test experiment, which is specifically as follows:
s1, adding the frozen tissue into a PBS buffer solution, shearing, settling and cleaning for 2 times, and removing a supernatant to obtain a first precipitate;
s2, adding 2ml of precooled lysate into the first precipitate, and performing pre-lysis on ice for 2min; homogenizing with Dounce homogenizer, and gently blowing and beating with a pipette up and down for 5-6 times to obtain homogenate; wherein the lysate is obtained by mixing 1ml EZ lysate (NUC-101, sigma-Aldrich) and 1ml PBS;
s3, transferring the homogenate to ice for incubation for 6min, adding 2ml of pre-cooled 4-percent BSA solution, blowing and beating for 6-8 times by using a Pasteur pipette, and stopping the reaction to obtain a first mixture;
s4, centrifuging the first mixture at the temperature of 4 ℃ for 5min by 300g, and removing a supernatant to obtain a second precipitate;
s5, resuspending the second pellet with 2ml of lysis solution and 4% BSA and incubating on ice for 3min to obtain a second mixture; wherein, the lysis solution: bovine serum albumin solution volume ratio = 125;
s6, removing fragments in the second mixture by using a flow sorting instrument to obtain a third precipitate; the method comprises the following specific steps:
s01, precooling a sample chamber and a sorting and collecting device of a BD FACSAria II type cell sorter to 4 ℃;
s02, filtering 200 mu l of the second mixture to obtain filtered cell nuclei, transferring the cell nuclei into a flow tube, and taking a filter cap of the flow tube as a non-staining control, namely a baseline for cell nucleus sorting;
s03, dyeing the remaining second mixture with propidium iodide, and then placing on ice for 10min; wherein the final concentration of propidium iodide is 10 μ g/ml;
s04, sorting the second mixture, wherein the sorter uses a 70nm nozzle with a default pressure of 20psi; exciting propidium iodide with a 488nm laser, and detecting by using a 600nm high-pass filter and a 610/20nm band-pass filter; collecting the sorted cell nuclei, placing the cell nuclei in a 15ml conical tube, adding 1ml PBS, and placing the cell nuclei on ice;
s05, centrifuging at the rotation speed of 1000g for 10min at 4 ℃ after sorting is finished; removing supernatant, adding 3ml PBS, and resuspending cell nucleus, wherein the resuspension method is a gentle spin method; then centrifuging the mixture for 10min at the rotating speed of 1000g at the temperature of 4 ℃; removing supernatant, adding 30-100 μ l PBS, further resuspending cell nucleus to obtain third precipitate
S7, the pellet was resuspended in 4ml of lysis buffer and 4% BSA buffer and incubated on ice for 5min. Centrifuging at 4 deg.C for 5min at 300g, and removing supernatant to obtain cell nucleus precipitate; wherein, the lysis solution: bovine serum albumin solution volume ratio = 125;
s8, resuspending the cell nucleus precipitate by using a second buffer solution, sieving the cell nucleus precipitate by using a 20um cell sieve (43-50020-50, pluraSelect), collecting the effluent cell suspension, and staining the cell nucleus suspension, wherein the picture is shown in figure 1;
s9, measuring and counting the concentration by adopting a Countess II automatic cell counter, wherein the cell nucleus concentration is 1152 nucleis/ul, and the total number of the cell nuclei is 10.2 ten thousand;
s10, capturing the cell nucleus suspension, marking a transcript in the cell nucleus, carrying out reverse transcription and constructing a library through a 10x Genomics single cell platform (according to the 10x Genomics marking and library construction experimental process), and expecting to capture 1 ten thousand cell nuclei;
s11, after the library construction is completed, adopting Illumina Novaseq TM 6000, sequencing, wherein the sequencing strategy is PE150, and the predicted output data volume is 100G Base; data analysis was performed based on cellrange and seruat software, with quality testing results shown in figure 2 and cell identification results shown in figure 3.
Example 3
In this example, single cell nuclear sequencing was performed on multiple organ frozen tissues (200 samples) such as brain, heart, liver, lung, kidney, intestine, and skeletal muscle of human, rat, mouse, drosophila, and pig species using the single cell nuclear sequencing experimental method (snRNA-seq) suitable for frozen tissues provided in example 1, and the results are shown in FIGS. 4-7; the corresponding fresh tissue (200 samples) was also sequenced using the classical single cell sequencing method (scRNA-seq), and the results are shown in FIGS. 4-7. The classical single cell sequencing method is shown in FIG. 8.
As can be seen from FIGS. 4 to 7, the single-cell nuclear sequencing assay (snRNA-seq) of the present invention has better high quality cell number, median of cell gene, total number of detected genes, and the like, and is comparable to the fresh tissue dissociation assay (scRNA-seq) in terms of the number of captured cells/cell nuclei, median of gene, total detected gene, and the like.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (7)
1. A method for preparing single cell nuclei of frozen tissue, comprising the steps of:
s1, adding frozen tissues into a first buffer solution for cleaning, shearing, settling and cleaning, and removing a supernatant to obtain a first precipitate;
s2, adding a precooled lysate into the first precipitate, performing pre-lysis on ice, and then homogenizing by using a homogenizer to obtain homogenate;
s3, transferring the homogenate to ice for incubation, adding a precooled bovine serum albumin solution, blowing and beating for 6-8 times by using a Pasteur pipette, and stopping reaction to obtain a first mixture; wherein the incubation time is 4-8min;
s4, centrifuging the first mixture at the temperature of 4 ℃ for 5min by 300g, and removing a supernatant to obtain a second precipitate;
s5, resuspending the second precipitate by using a lysate and a bovine serum albumin solution, and incubating for 2-4min on ice to obtain a second mixture;
s6, removing fragments in the second mixture by using a flow sorting instrument to obtain a third precipitate;
s7, adding lysis solution and BSA solution into the third precipitate for resuspension, incubating on ice for 5min, centrifuging at 300g at 4 ℃ for 5min, and removing supernatant to obtain cell nucleus precipitate;
and S8, adding a second buffer solution for resuspending the cell nucleus sediment into the cell nucleus sediment for resuspension, sieving the cell nucleus sediment by using a 20-micron cell sieve, and collecting an effluent cell suspension, namely the single cell nucleus of the frozen tissue.
2. The method for preparing single cell nuclei of frozen tissue according to claim 1, wherein the specific process of step S6 is as follows:
s61, precooling a sample chamber and a sorting and collecting device of a cell sorter to 4 ℃;
s62, filtering 200 mu l of the second mixture to obtain filtered cell nuclei, transferring the cell nuclei into a flow tube, and taking a filter cap of the cell nuclei as a non-staining control, namely as a baseline for cell nucleus sorting;
s63, dyeing the remaining second mixture with propidium iodide, and then placing on ice for 10min;
s64, sorting the second mixture, wherein the sorter uses a 70nm nozzle with a default pressure of 20psi; exciting propidium iodide with a 488nm laser, and detecting by using a 600nm high-pass filter and a 610/20nm band-pass filter; collecting the sorted cell nuclei, placing the cell nuclei in a 15ml conical tube, adding 1ml PBS, and placing the cell nuclei on ice;
s65, centrifuging at the rotating speed of 1000g for 10min at 4 ℃ after sorting is finished; removing supernatant, adding 3ml PBS, and resuspending cell nucleus, wherein the resuspension method is a gentle spin method; then centrifuging the mixture for 10min at the rotating speed of 1000g at the temperature of 4 ℃; after removing the supernatant, 30-100. Mu.l of PBS was added, and the nuclei were further resuspended to obtain a first nucleus pellet.
3. A method for preparing a single cell nucleus of frozen tissue according to claim 1, wherein in step S1, the first buffer solution is PBS.
4. A method for the preparation of single cell nuclei of frozen tissue according to claim 1, characterized in that the lysate is a mixture of EZ lysate and PBS; wherein the volume ratio of the EZ lysate to the PBS is 1:1.
5. the method of claim 1, wherein the second buffer comprises 1 x PBS, 0.07wt% BSA and 0.1wt% RNase inhibitor.
6. A method for preparing single cell nuclei of frozen tissue according to claim 1, characterized in that the frozen tissue is a frozen tissue sample of an animal species.
7. Use of the method of claim 1 for single cell nuclear sequencing of fresh, large cell containing or difficult to dissociate tissue.
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