CN114350610A - Kit for separating brain and spinal cord tissue cell nucleus and cell nucleus separation method - Google Patents
Kit for separating brain and spinal cord tissue cell nucleus and cell nucleus separation method Download PDFInfo
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Abstract
The invention relates to the technical field of single cell nucleus transcriptome sequencing, in particular to a kit for separating cell nuclei of brain and spinal cord tissues and a cell nucleus separation method. The kit provided by the invention comprises lysis solution and cell nucleus cleaning solution. The invention also provides a method for extracting cell nucleus by using the kit. The kit and the extraction method only need a small amount of tissues for extraction, and can obtain the mononuclear cell nucleus suspension with higher mononuclear cell nucleus quantity and quality without complex flow cell sorting, can well meet the requirement of sequencing of mononuclear cell nucleus transcriptome, obtain high-quality transcriptome data, and have the advantages of low cost and simple and rapid operation.
Description
Technical Field
The invention relates to the technical field of single cell nucleus transcriptome sequencing, in particular to a kit for separating cell nuclei of brain and spinal cord tissues and a cell nucleus separation method.
Background
In recent years, sequencing of single cell nuclear transcription sets has attracted more and more attention because of the characteristics that the sequencing has no requirement on cell diameter and has no dissociation effect, and can truly reflect cell types of solid tissues and the like. In single cell nuclear sequencing, it is first necessary to isolate a relatively high quality suspension of single cell nuclei. In order to meet the requirement of sequencing of the single cell nucleus transcriptome, most of the currently common single cell nucleus separation methods need flow type separation and purification of cell nuclei, the operation of flow type separation is complicated, the cost is high, and the requirement on the tissue quantity is high due to more cell nuclei lost by flow type separation, and the quantity is generally 200mg (about the size of soybean). In addition, at present, no complete quality control standard exists for the cell nucleus obtained by flow sorting, and sequencing is directly performed after flow sorting, which may cause poor data and needs repeated experiments.
Disclosure of Invention
One of the objects of the present invention is to provide a kit for isolating nuclei from brain and spinal cord tissue of animals. It is another object of the present invention to provide a method for separating animal brain and spinal cord tissue nuclei, which is applicable to extraction of nuclei for single-nucleus transcriptome sequencing.
Sequencing of the single-nucleus transcriptome has high requirement on the integrity of the nucleus, and requires less impurities of the nucleus, no obvious fragments and low agglomeration rate. The invention finds that the nuclear membrane of the cell nucleus of the brain and the spinal cord is easier to break compared with other tissues, and the complexity of the brain and the spinal cord tissues causes more impurities of the cell nucleus obtained by extraction, and the cell nucleus needs to be purified by adopting flow sorting, but the loss of the cell nucleus in the flow sorting process is more, and the flow cell sorter is expensive and has higher operation requirement. The method for extracting the cell nucleus from other tissues in the prior art has the problems of low cell membrane lysis rate and low cell nucleus extraction amount, or has the problems of poor integrity, more impurities and high agglomeration rate of the extracted cell nucleus, and the problems can generate obvious adverse effects on the sequencing effect of the single cell nucleus transcription group.
The invention aims to develop a cell nucleus separation and extraction method suitable for sequencing of the single cell nucleus transcriptome of brain and spinal cord tissues, and continuously optimizes and verifies the reagent composition and the separation method used in the separation process of the cell nucleus according to the characteristics of the cells and the cell nucleus of the brain and spinal cord tissues, thereby improving the quality of the cell nucleus and well meeting the requirement of sequencing of the single cell nucleus transcriptome.
Specifically, the invention provides the following technical scheme:
firstly, the invention provides a kit for separating brain and spinal cord tissue cell nuclei, which comprises lysate and cell nucleus cleaning solution; the cell nucleus cleaning solution takes a DMEM culture medium as a solvent and comprises the following components: 8-12% FBS, 0.2-1U/. mu.L RNase inhibitor.
In the separation process of cell nucleus, each separation step after cell lysis all involves the use of cell nucleus cleaning solution, the effect of which mainly lies in cleaning and suspending cell nucleus, and the cell cleaning solution is very important for the integrity of cell nucleus membrane and the discreteness of cell nucleus. In research and development, compared with the method of compounding water or other buffer solutions and protein, the method has the advantages that the DMEM culture medium is used as the solvent of the cell nucleus cleaning solution, and meanwhile, the FBS with a certain concentration is added, so that the breakage of cell nuclei in the separation process can be obviously reduced, the discreteness of the cell nuclei is improved, and the separation effect of the cell nuclei is obviously improved.
Preferably, the cell nucleus cleaning solution takes a DMEM medium as a solvent and comprises the following components: 10-12% FBS, 0.2-1U/. mu.L RNase inhibitor.
The cell nucleus cleaning solution can effectively reduce the breakage of cell nuclei and improve the separation effect of the cell nuclei.
The lysis solution comprises the following components: 250-350mM sucrose, 5-25mM KCl, 3-8mM MgCl210-20mM Tris-HCl pH 7.0-7.5, 0.05-0.2% Nonidet P40 Substitee, 0.2-1U/. mu.L RNase inhibitor, 0.8-1.5mM DTT.
During the separation of the cell nucleus, the lysis solution acts to lyse the cell membrane, thereby releasing the cell nucleus. The sufficient cell membrane lysis is beneficial to release a large amount of cell nucleus, but the integrity and discreteness of the nuclear membrane should be ensured as much as possible during the lysis treatment, the impurities and the agglomeration rate are reduced, and meanwhile, the degradation of RNA in the cell nucleus needs to be reduced as much as possible.
The invention carries out a great deal of research on the composition of the lysis solution and finds that the lysis solution can well ensure the integrity of a nuclear membrane and reduce the generation of impurities while ensuring the lysis rate of a cell membrane, has better protection effect on RNA in the cell nucleus, and simultaneously has better discreteness on the separated cell nucleus. The combined use of sucrose and Nonidet P40 substitee in the above concentration range in the above lysate is particularly important for the achievement of the above effect, and the removal thereof or the replacement or change of the concentration with other functionally similar components can have a significant adverse effect on the achievement of the above effect.
Preferably, the lysate takes a DMEM medium as a solvent and comprises the following components: 310-330mM sucrose, 10-25mM KCl, 4-6mM MgCl210-12mM Tris-HCl pH 7.0-7.5, 0.1-0.15% Nonidet P40 Substitee, 0.2-1U/. mu.L RNase inhibitor, 0.8-1.2mM DTT.
The invention finds that the lysis solution takes the DMEM culture medium as the solvent, so that the integrity of the cell nucleus membrane is better ensured, the generation of impurities is reduced, the lysis effect is ensured, and the separation effect of the cell nucleus is obviously reduced by adopting water or other buffer solutions to replace the DMEM culture medium.
In a preferred embodiment of the present invention, the lysis solution uses DMEM as a solvent, and comprises the following components: 320mM sucrose, 25mM KCl, 5mM MgCl210mM Tris-HCl pH 7.5, 0.1% Nonidet P40 Substitee, 0.2U/. mu.L RNase inhibitor, 1mM DTT.
When carrying out the cell nucleus separation and extracting, adopt above-mentioned lysate can guarantee higher cell membrane schizolysis efficiency, improve the yield of cell nucleus, simultaneously, guarantee the integrality of nuclear membrane well, reduce impurity and produce, reduce the degradation of cell nucleus RNA, reduce the agglomeration of cell nucleus, greatly reduced the degree of difficulty of follow-up purification process for no longer rely on flow cell to select separately also can obtain the cell nucleus of higher quality.
The DMEM culture medium in the lysis solution and the cell nucleus cleaning solution is preferably a low-sugar DMEM culture medium.
The kit may also contain other reagents required for nuclear separation.
Based on the kit, the invention provides a method for separating nuclei of brain and spinal cord tissues, which adopts the kit to separate the nuclei and comprises the following steps: carrying out lysis treatment on a sample to be treated in lysis solution to obtain a lysis product, mixing the lysis product with the cell nucleus cleaning solution, carrying out first filtration and solid-liquid separation to obtain a cell nucleus crude extract, and carrying out the following treatment (1) or (2) on the cell nucleus crude extract:
(1) mixing the cell nucleus crude extract with the cell nucleus cleaning solution, carrying out second filtration, then carrying out solid-liquid separation, and collecting cell nucleus precipitates;
(2) mixing the cell nucleus crude extract with the cell nucleus cleaning solution to obtain cell nucleus suspension, mixing the cell nucleus suspension with a density gradient reagent, performing density gradient centrifugation, and collecting precipitates; and mixing the precipitate with the cell nucleus cleaning solution, carrying out second filtration, carrying out solid-liquid separation, and collecting the cell nucleus precipitate.
The treatment in (2) is suitable for the case that after the first filtration, the impurities are relatively more or the requirement on the purity of the cell nucleus is relatively higher, and if the impurities are less after the first filtration, the treatment in (1) can be directly carried out.
In the above-described method, the amount of the lysis solution is preferably (10 to 30). mu.L/mg of the sample to be treated.
Mixing the lysate and the cell nucleus wash solution in the ratio of (1-2): 1.
when the above lysis solution is used, the preferred conditions for the lysis treatment are: treating at 0-4 deg.C for 3-6 min.
The cracking treatment comprises the following steps: firstly, a sample to be processed is cut into pieces in a lysis solution, and then incubation is carried out.
Compared with the modes of grinding and the like, the tissue crushing mode by using the scissors can ensure the cracking efficiency and is beneficial to ensuring the integrity of the cell nuclear membrane.
Preferably, the density gradient centrifugation is performed by mixing the nuclear suspension with a density gradient reagent in a volume ratio of about (3.5-3.8): 1, and then centrifuged at 2800-.
The total volume of the sucrose density gradient centrifugation system is controlled within 2mL, so that the requirement of single nucleus transcriptome sequencing on the number of nuclei can be met.
In the method for separating cell nuclei described above, the first filtration is filtration through a 70 μm filter. The second filtration was filtration through a 40 μm filter membrane.
By adopting the method for separating the cell nucleus, the requirement of single cell nucleus transcriptome sequencing on the number of the cell nucleus can be met only by a very small amount of tissues.
As a preferred embodiment of the present invention, the method for separating brain and spinal cord tissue nuclei comprises the steps of:
(1) taking 20-50mg of tissue to be treated, adding 50-150 mu L of lysis solution, starting timing, shearing the tissue into small pieces, then adding 350-450 mu L of lysis solution, cracking at 0-4 ℃, and carrying out cracking treatment for 4-6min from the start timing;
(2) after the cracking is finished, adding 500 mu L of cell nucleus cleaning fluid to stop the cracking to obtain a cracking product;
(3) filtering the cracked product with 70 μm filter membrane, and centrifuging at 4 deg.C and 500g for 4-6 min;
(4) removing supernatant after centrifugation, resuspending the precipitate (crude extract of cell nucleus) with cell nucleus cleaning solution, filtering with 40 μm small filter membrane, centrifuging at 4 deg.C and 500g for 4-6min with horizontal centrifuge, removing supernatant after centrifugation, and resuspending the precipitate with cell nucleus cleaning solution.
As another preferred embodiment of the present invention, the method for separating brain and spinal cord tissue nuclei comprises the steps of:
(1) taking 20-50mg of tissue to be treated, adding 50-150 mu L of lysis solution, starting timing, shearing the tissue into small pieces, then adding 350-450 mu L of lysis solution, cracking at 4 ℃, and carrying out cracking treatment for 4-6min from the start timing;
(2) after the cracking is finished, adding 500 mu L of cell nucleus cleaning fluid to stop the cracking to obtain a cracking product;
(3) filtering the cracked product with 70 μm filter membrane, and centrifuging at 4 deg.C and 500g for 4-6 min;
(4) removing the supernatant after the centrifugation is finished to obtain a cell nucleus crude extract, and adding 775 mu L of cell nucleus cleaning solution to resuspend the cell nucleus crude extract to obtain a cell nucleus suspension; 215 mu L of American and whirly density gradient reagent (the goods number: 130-; centrifuging at 4 deg.C and 3000g for 8-12min, wherein the lifting acceleration of the centrifuge is 1;
(5) removing impurities in the middle layer after centrifugation, then sucking the supernatant, resuspending the precipitate with a cell nucleus cleaning solution, filtering with a 40-micron filter membrane, centrifuging at 500g for 4-6min at 4 ℃, removing the supernatant, and then resuspending the precipitate with a cell nucleus cleaning solution to obtain the separated cell nucleus.
Furthermore, the invention provides a single cell nucleus transcriptome sequencing method, which is characterized in that the method for separating the cell nucleus of the brain and spinal cord tissue is adopted to separate the cell nucleus in a sample to be sequenced, and then the single cell nucleus transcriptome sequencing is carried out on the cell nucleus.
In the practice of sequencing a large number of single-cell nucleus transcriptome, the quality control standard of the cell nucleus for sequencing is found, and the efficiency and the data quality of sequencing the single-cell nucleus transcriptome can be obviously improved by using the cell nucleus meeting the quality control standard.
The quality control criteria for the nuclei were as follows: the cell nucleus concentration is 700-1200/mu L, the cell activity is lower than 5%, the agglomeration rate is lower than 15%, the volume is not lower than 60 mu L, at least 80% of the cell nucleus membrane is complete, and the cell nucleus membrane is free from rupture and obvious impurities and fragments.
The quality control standard can adopt a microscope and a blood counting chamber to carry out the quality inspection of the integrity of the cell nucleus, and a countstar cell counter is used for counting.
By means of the technical scheme, the invention at least has the following beneficial effects:
1. in the kit for separating the brain and spinal cord tissue cell nucleus, the cell nucleus cleaning solution can reduce the breakage of the cell nucleus, the lysate has higher cell membrane lysis rate, the cooperation of the cell nucleus cleaning solution and the lysate can well ensure the integrity of the nuclear membrane, reduce the generation of impurities and the degradation of RNA, and can reduce the agglomeration rate of the cell nucleus.
2. The cell nucleus separation method only needs a small amount of tissue for extraction, and can obtain the single cell nucleus suspension with higher cell nucleus quantity and quality without complicated flow cell sorting.
3. The cell membrane cracking rate of the cell nucleus separation method is high, the prepared cell nucleus is low in cell activity and agglomeration rate, the integrity of the cell nucleus is high, cell fragments and impurities are few, RNA in the cell nucleus is degraded, the obtained gene number is high, the requirement of sequencing of a single cell nucleus transcriptome can be well met, and high-quality transcriptome data can be obtained.
4. The method for separating the cell nucleus does not need expensive instruments such as a flow cytometer and the like, and has the advantages of low cost, simple and quick operation and short required time (the preparation of the cell nucleus can be completed in about 40 minutes).
5. The invention establishes the quality control standard of cell nucleus meeting the sequencing requirement of the single cell nucleus transcriptome, the quality control standard can obtain high-quality transcriptome data, and the cell nucleus prepared by the cell nucleus separation method can meet the quality control standard.
Drawings
FIG. 1 shows the result of quality control of mouse hypothalamic nucleus microscope in example 1 of the present invention.
FIG. 2 shows the quality control results of the mouse spinal cord nucleus microscope in example 2 of the present invention.
FIG. 3 shows the quality control results of the mouse brain nuclei microscope of example 3 of the present invention, wherein A is the result of scheme 1 and B is the result of scheme 2.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The Nonidet P40 subcostinate used in the following examples was purchased from Sigma under the cat number 74385; the RNase inhibitor used was Sigma detector RNase inhibitor, purchased from Sigma, cat # 3335402001; DTT was purchased from Sigma under cat # 646563; 70 μm filters were purchased from Bel-Art under the cat number H13680-0070; the 40 μm filter membrane was purchased from Bel-Art, cat # H13680-0040; the density gradient reagent is a reagent which is whirly in America, and the goods number is 130-; DMEM (low sugar) medium is an industrial reagent, and the product number is 600008-.
Example 1 isolation of mouse hypothalamic nuclei
The present embodiment provides a method for separating cell nuclei by using mouse hypothalamic tissue as an experimental material, which comprises the following specific steps:
(1) frozen 20mg mouse hypothalamus was immediately transferred to a 1.5mL pre-cooled centrifuge tube; adding 100 μ L of lysis solution, timing immediately, cutting into small pieces with precooled scissors, wherein the smaller the size is, the better the size is, the cutting time is about 2 min; continuously adding 400 μ L of lysis solution, and performing total lysis on ice for 5min (from the beginning of adding lysis solution for the first time to the end of lysis);
the formula of the 500 mu L cell nucleus lysate is as follows: 320mM sucralose, 25mM KCl, 5mM MgCl210mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 substute, 0.2U/. mu.L Sigma Detector RNase inhibitor, 1mM DTT, DMEM to 500. mu.L;
(2) after the cracking is finished, stopping the cracking by using 500 mu L of cell nucleus cleaning fluid to obtain a cracking product;
the formula of the cell nucleus cleaning solution is as follows: DMEM is used as a solvent and contains 10% FBS and 0.2U/. mu.L Sigma Detector RNase inhibitor;
(3) filtering the lysate with a pipette-type 70 μm filter membrane into a new 1.5mL precooled centrifuge tube, and centrifuging at 500g for 5min at 4 ℃ by using a horizontal centrifuge;
(4) removing supernatant after centrifugation, resuspending the precipitate with 1mL of cell nucleus cleaning solution, and filtering with a 40 μm small filter membrane into a new 1.5mL precooled centrifuge tube; centrifuging at 500g for 5min at 4 deg.C with horizontal centrifuge, removing supernatant after centrifugation, and resuspending the precipitate with 60 μ L cell nucleus cleaning solution to obtain separated cell nucleus.
Nuclear integrity quality control was performed using a microscope (fig. 1), and counted using a countstar cell counter; the concentration of the cell nucleus is adjusted to 700-.
Example 2 isolation of mouse spinal cord nuclei
In this embodiment, a method for separating cell nuclei is provided by using mouse spinal cord tissue as an experimental material, and the specific steps are as follows:
(1) taking frozen mouse spinal cord tissue of 50mg, and immediately transferring the mouse spinal cord tissue into a 1.5mL precooled centrifuge tube; adding 100 μ L of lysis solution, timing immediately, cutting into small pieces with precooled scissors, wherein the smaller the size is, the better the size is, the cutting time is about 2 min; continuously adding 400 μ L of lysis solution, and performing total lysis on ice for 5min (from the beginning of adding lysis solution for the first time to the end of lysis);
the formulation of 500 μ L of cell nuclear lysate is as follows: 320mM sucralose, 25mM KCl, 5mM MgCl210mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 substute, 0.2U/. mu.L Sigma Detector RNase inhibitor, 1mM DTT, DMEM to 500. mu.L.
(2) After the cracking is finished, stopping the cracking by using 500 mu L of cell nucleus cleaning fluid to obtain a cracking product;
the formula of the cell nucleus cleaning solution is as follows: DMEM is used as a solvent and contains 10% of FBS and 0.2U/. mu.L of Sigma Detector RNase inhibitor;
(3) filtering the lysate with a suction head type 70 μm filter membrane into a new 1.5mL precooled centrifuge tube, and centrifuging at 500g for 5min at 4 ℃ by using a horizontal centrifuge;
(4) removing the supernatant after the centrifugation is finished to obtain a cell nucleus crude extract, and adding 775 mu L of cell nucleus cleaning solution to resuspend the cell nucleus crude extract to obtain a cell nucleus suspension; adding 215 mu L of American and natural and gentle reagent (the goods number: 130-; then 1mL of cell nucleus cleaning solution is carefully paved along the tube wall of the centrifuge tube; centrifuging at 4 deg.C for 10min at 3000g, with the lifting acceleration of the centrifuge being 1;
(5) removing impurities in the middle layer after centrifugation, then sucking the supernatant, resuspending the precipitate with 1mL of cell nucleus cleaning solution, filtering with a 40 μm filter membrane, centrifuging at 4 deg.C and 500g for 4-6min, removing the supernatant, and resuspending the precipitate with 60 μ L of cell nucleus cleaning solution to obtain separated cell nucleus.
Performing quality inspection of the integrity of the cell nucleus of the high-concentration cell nucleus under a microscope (figure 2), and counting by using a countstar cell counter; adjusting the concentration of the cell nucleus to 700-1200/muL according to the counting concentration, wherein the final concentration is 1010/muL and the volume is 60 muL; the cell activity is 0%, the agglomeration rate is 2.92%, 80% of cell nucleus membranes are complete, and no rupture, obvious impurities and fragments exist.
Example 3
The embodiment provides a method for separating cell nuclei by taking mouse brain tissue as an experimental material, which comprises the following specific steps:
(1) frozen 50mg mouse 6-week brain tissue was taken and immediately transferred to a 1.5mL pre-cooled centrifuge tube; adding 100 μ L of lysis solution, timing immediately, cutting into small pieces with precooled scissors, wherein the smaller the size is, the better the size is, the cutting time is about 2 min; continuously adding 400 μ L of lysis solution, and performing total lysis on ice for 5min (from the beginning of adding lysis solution for the first time to the end of lysis);
the formulation of 500 μ L of cell nuclear lysate is as follows: 320mM sucralose, 25mM KCl, 5mM MgCl210mM Tris-HCl (pH 7.5), 0.1% Nonidet P40 subcostute, 0.2U/. mu.L Sigma Detector RNase inhibitor, 1mM DTT, in solvent make-up to 500. mu.L, wherein scheme 1 is PBS as solvent and scheme 2 is DMEM medium as solvent.
(2) After the cracking is finished, stopping the cracking by using 500 mu L of cell nucleus cleaning fluid to obtain a cracking product;
scheme 1: the formula of the cell nucleus cleaning solution is as follows: PBS is used as a solvent, and the solution contains 1% BSA and 0.2U/. mu.L Sigma Detector RNase inhibitor;
scheme 2: the formula of the cell nucleus cleaning solution is as follows: DMEM is used as a solvent and contains 10% of FBS and 0.2U/. mu.L of Sigma Detector RNase inhibitor;
(3) filtering the lysate with a suction head type 70 μm filter membrane into a new 1.5mL precooled centrifuge tube, and centrifuging at 500g for 5min at 4 ℃ by using a horizontal centrifuge;
(4) after centrifugation, the supernatant was aspirated, the pellet was resuspended in 1mL of cell nucleus wash (formula for cell nucleus wash in protocols 1 and 2, supra), filtered through a 40 μm filter, centrifuged at 500g for 4-6min at 4 deg.C, and after removal of the supernatant, the pellet was resuspended in 60 μ L of cell nucleus wash to obtain separated cell nuclei.
The high concentration nuclei were examined microscopically for nuclear integrity and counted using a countstar cell counter.
The microscopic examination results of the cell nuclei isolated in scheme 1 and scheme 2 are shown in a of fig. 3 and B of fig. 3, respectively.
Adjusting the concentration of the cell nucleus to 700-; the final concentration of nuclei obtained in scheme 1 was 1360/. mu.L in a volume of 60. mu.L; the cell activity is 1.27%, the agglomeration rate is 3.39%, part of cell nucleus is broken, and 70% of cell nucleus membrane is complete; the final concentration of nuclei in protocol 2 was 1400/μ L in a volume of 60 μ L; the cell activity is 0 percent, the agglomeration rate is 4.06 percent, the nuclear membrane is basically not broken, and more than 80 percent of the nuclear membrane is complete.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A kit for separating brain and spinal cord tissue cell nuclei is characterized by comprising a lysate and a cell nucleus cleaning solution; the cell nucleus cleaning solution takes a DMEM culture medium as a solvent and comprises the following components: 8-12% FBS, 0.2-1U/. mu.L RNase inhibitor.
2. The kit of claim 1, wherein the cell nucleus washing solution uses a DMEM medium as a solvent and comprises the following components: 10-12% FBS, 0.2-1U/. mu.LRNA enzyme inhibitor.
3. The kit according to claim 1 or 2, characterized in that the lysis solution comprises the following components: 250-350mM sucrose, 5-25mM KCl, 3-8mM MgCl210-20mM Tris-HCl pH 7.0-7.5, 0.05-0.2% Nonidet P40 Substitee, 0.2-1U/. mu.L RNase inhibitionAgent, 0.8-1.5mM DTT.
4. The kit according to claim 3, wherein the lysate is in DMEM medium as a solvent and comprises the following components: 310-330mM sucrose, 10-25mM KCl, 4-6mM MgCl210-12mM Tris-HCl pH 7.0-7.5, 0.1-0.15% Nonidet P40 Substitee, 0.2-1U/. mu.L RNase inhibitor, 0.8-1.2mM DTT.
5. A method for separating nuclei from brain and spinal cord tissue, comprising the steps of using the kit of any one of claims 1 to 4 for separation of nuclei: carrying out lysis treatment on a sample to be treated in the lysis solution to obtain a lysis product, mixing the lysis product with the cell nucleus cleaning solution, carrying out first filtration and solid-liquid separation to obtain a cell nucleus crude extract, and carrying out the following treatment (1) or (2) on the cell nucleus crude extract:
(1) mixing the cell nucleus crude extract with the cell nucleus cleaning solution, carrying out second filtration, then carrying out solid-liquid separation, and collecting cell nucleus precipitates;
(2) mixing the cell nucleus crude extract with the cell nucleus cleaning solution to obtain cell nucleus suspension, mixing the cell nucleus suspension with a density gradient reagent, performing density gradient centrifugation, and collecting precipitates; and mixing the precipitate with the cell nucleus cleaning solution, carrying out second filtration, carrying out solid-liquid separation, and collecting the cell nucleus precipitate.
6. The method for separating brain and spinal cord tissue nuclei of claim 5, wherein the lysis treatment is treatment at 0-4 ℃ for 3-6 min.
7. The method for separating brain and spinal cord tissue nuclei of claim 5 or 6, wherein in the lysis treatment, the amount of the lysis solution is (10-30) μ L/mg of the sample to be treated;
preferably, the ratio of the lysate to the cell nucleus wash is (1-2): 1.
8. the method for separating brain and spinal cord tissue nuclei of any one of claims 5 to 7, wherein the density gradient centrifugation is performed by mixing the cell nucleus suspension and a density gradient reagent in a volume ratio of (3.5-3.8): 1, and then centrifuged at 2800-.
9. The method for separating brain and spinal cord tissue nuclei of any one of claims 5 to 8, wherein the first filtration is filtration through a 70 μm filter;
and/or, the second filtration is filtration through a 40 μm filter membrane.
10. A single cell nuclear transcriptome sequencing method is characterized in that the method for separating brain and spinal cord tissue cell nuclei of any one of claims 5 to 9 is adopted to separate cell nuclei in a sample to be sequenced, and the cell nuclei are subjected to single cell nuclear transcriptome sequencing;
preferably, the concentration of the cell nucleus is 700-.
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| CN109628555A (en) * | 2019-01-30 | 2019-04-16 | 杭州瑞普基因科技有限公司 | People suitable for unicellular sequencing freezes tumor tissue cell's core separation method |
| CN112280828A (en) * | 2020-10-22 | 2021-01-29 | 上海交通大学医学院 | In vitro tissue cell nucleus separation method for reducing single cell amplification bias |
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| US20210270703A1 (en) * | 2020-02-28 | 2021-09-02 | 10X Genomics, Inc. | Method for isolating nuclei and cells from tissues |
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| CN109628555A (en) * | 2019-01-30 | 2019-04-16 | 杭州瑞普基因科技有限公司 | People suitable for unicellular sequencing freezes tumor tissue cell's core separation method |
| US20210270703A1 (en) * | 2020-02-28 | 2021-09-02 | 10X Genomics, Inc. | Method for isolating nuclei and cells from tissues |
| CN112280828A (en) * | 2020-10-22 | 2021-01-29 | 上海交通大学医学院 | In vitro tissue cell nucleus separation method for reducing single cell amplification bias |
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