Kit for separating hepatocyte nucleuses and method for separating hepatocyte nucleuses

Abstract

The invention relates to an animal tissue cell nucleus extraction method, and belongs to the technical field of cell nucleus extraction. The invention discloses a kit for separating liver tissue cell nucleuses and a separation method. The kit provided by the invention not only comprises a lysis solution, a cleaning and resuspending solution and a fragment removing component, but also provides an extraction method suitable for the kit. By utilizing the method, the cell nucleuses can be extracted from a small amount of tissues, special instruments and equipment are not needed, and the extraction of the cell nucleuses can be completed by utilizing conventional instruments such as a centrifugal machine and a microscope. The kit and the extraction method for extracting the cell nucleus have the advantages of high cell nucleus yield, few impurities, simplicity in operation, short treatment time, low cost and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of single-cell nuclear transcriptome sequencing, and particularly to a kit and a method for isolating liver tissue cell nuclei. Background Art

[0002] In recent years, single-cell sequencing technology has been widely applied. However, liver parenchymal cells are very fragile, prone to rupture, and have a high mRNA content, which easily leads to excessive environmental mRNA and abnormal single-cell sequencing. At the same time, tissue dissociation will result in the loss of a large number of fragile cells, thus causing a large change in the proportion of original cells in the tissue, resulting in the inability of single-cell sequencing to truly reflect tissue characteristics. Single-cell nuclear transcriptome sequencing has attracted much attention because it has no excessive requirements for cell size and stability, and does not have the characteristic of losing some cells due to tissue dissociation, and can more truly reflect the cell types of the original tissue. Isolating high-quality single cell nuclei is a prerequisite for single-cell nuclear sequencing. To obtain a cell nucleus suspension that can meet the requirements of single-cell nuclear transcriptome sequencing, currently, most methods are to lyse the tissue and then purify it by flow sorting to obtain high-purity cell nuclei. However, flow sorting requires special instruments, professional sorting personnel, high experimental costs, large cell nucleus losses, and high requirements for tissue volume. Nuclei extraction requires more than 150 mg of tissue, and the tissue volume available in the experiment often cannot meet the requirements. Summary of the Invention

[0003] The present invention discloses a kit and a method for isolating cell nuclei from animal liver tissue. Another object of the present invention is to provide a method for isolating cell nuclei from animal liver tissue, but not limited to animal liver tissue samples. The cell nuclei extracted by this method are suitable for single-cell nuclear transcriptome sequencing.

[0004] Single-cell nuclear transcriptome sequencing has high requirements for the quality of cell nuclei. It not only requires high cell nucleus integrity but also requires few impurities and a low aggregation rate in the cell nucleus suspension. When extracting cell nuclei from animal tissues, it is found that the cell nuclei extracted from liver tissue are relatively fragile and prone to deformation and rupture, resulting in a high content of free mRNA in the environment, affecting the quality of single-cell nuclear sequencing, and the cell nuclei are prone to aggregation, increasing the risk of subsequent single-cell capture and pore blockage. A large amount of impurities will also remain in the tissue when extracting cell nuclei, and a flow cytometer is needed for purification, but the recovery rate of cell nuclei treated by this method is low and the sorting cost is relatively high. Existing methods for extracting cell nuclei from other tissues have a series of problems, such as: poor cell nucleus stability, prone to rupture; unsatisfactory tissue lysis effect, low cell nucleus yield; more impurities, unable to be removed, etc. These problems will have an adverse impact on subsequent single-cell nuclear transcriptome sequencing.

[0005] The present invention is applicable to the isolation and extraction of liver tissue cell nuclei. The cell nuclei extracted by this method can be used for single-cell nucleus transcriptome sequencing. During the development process, the reagent formulations and experimental methods for tissue lysis, debris removal, and cell nucleus washing and resuspension were continuously optimized, greatly improving the cell nucleus yield, stability, and debris removal efficiency, providing high-quality cell nuclei for subsequent cell nucleus transcriptome sequencing.

[0006] First, the present invention is applicable to the isolation of liver tissue cell nuclei, which includes lysis buffer LB Buffer, washing and resuspension buffer WR Buffer, and debris removal components (B1 / B2 / B3).

[0007] The selection of surfactants and buffers plays a crucial role in tissue lysis and maintaining the stability of cell nuclei. The lysis buffer LB Buffer of the present invention uses D-Hank's buffer as the solvent. By optimizing the concentration of the surfactant Nonidet P40 Substitute, and adding an appropriate amount of sucrose and magnesium ions to the lysis buffer, the stability of the cell nucleus product is significantly enhanced, ensuring the integrity of the cell nuclei.

[0008] The washing and resuspension buffer WR Buffer is based on D-Hank's buffer and contains: 1% BSA, DTT (1-2 mM), RNase inhibitor (0.2-1 U / μL), which can maintain the stability of cell nuclei, effectively reduce cell nucleus rupture and aggregation, and can be directly used for subsequent single-cell nucleus sequencing (it does not contain RNase inhibitors such as calcium ions and magnesium ions).

[0009] The tissue is ground and homogenized in the lysis buffer to make the cells contact the lysis buffer as much as possible. The lysis buffer lyses the cell membrane, thereby releasing the cell nuclei. Therefore, to release the cell nuclei from the tissue to the greatest extent, the tissue needs to be fully homogenized to rupture as many cell membranes as possible and fully release the cell nuclei. However, excessive homogenization and lysis are not conducive to the stability of the cell nuclei. Therefore, during the treatment process, the integrity and stability of the cell nuclei should also be ensured as much as possible. The grinding should not be excessive, and the lysis time should not be too long.

[0010] The present invention deeply optimizes the lysis buffer LB Buffer, and screens out D-Hank's as the best buffer. In addition, a certain amount of sucrose, magnesium ions, Tween-20 and BSA are added as aids, effectively improving the lysis efficiency of the lysis buffer and the stability of cell nuclei. When using this reagent to extract cell nuclei from tissues, not only can the tissues be fully lysed, with a high yield of cell nuclei, but it also has a certain protective effect on the nuclear membrane, can maintain the stability of the nuclear membrane, prevent rupture, and reduce nuclear aggregation. At the same time, adding an appropriate amount of RNase inhibitor and DTT can prevent the degradation of mRNA in the cell nuclei. Nonidet P40 Substitute in the lysis buffer LB Buffer plays a role in lysing the cell membrane, and its combined use with sucrose within the above concentration range can achieve the above effects. Removing sucrose or changing the sucrose concentration will affect the tissue lysis efficiency and the stability of cell nuclei.

[0011] The above lysis buffer LB Buffer uses D-Hank's buffer as the solvent, and in addition contains: 0.1% Tween-20, 1% BSA, 0.05% - 0.1% Nonidet P40 Substitute, 250 - 300 mM sucrose, 3 - 5 mM MgCl 2 ·6H 2 O, 0.2 - 1 U / μL RNase inhibitor, 1 - 2 mM DTT. The washing and resuspension buffer WR Buffer uses D-Hank's buffer as the solvent, and in addition contains: 1% BSA, 0.2 - 1 U / μL RNase inhibitor, 1 - 2 mM DTT.

[0012] The present invention discovers that using D-Hank's buffer as the solvent of the lysis buffer is more conducive to maintaining the integrity of the nuclear membrane, and at the same time does not affect the tissue lysis effect. However, using 1xPBS buffer, Tris buffer or water as the solvent will reduce the stability and separation effect of cell nuclei.

[0013] When separating cell nuclei from liver tissues, using the above lysis buffer LB Buffer can improve the cell membrane lysis efficiency and the yield of cell nuclei. At the same time, using the washing and resuspension buffer WR Buffer can well maintain the integrity of the nuclear membrane, prevent nuclear rupture, reduce nuclear clumping, and reduce the risk of RNA degradation in cell nuclei, and can quickly and efficiently obtain high-quality cell nuclei. At the same time, the cell nucleus suspension resuspended by the washing and resuspension buffer WR Buffer can be directly used for subsequent single-cell nucleus sequencing experiments.

[0014] The above kit contains other reagents required for cell nucleus separation, such as: lysis buffer LB Buffer, washing and resuspension buffer WR Buffer, debris removal components (B1 / B2 / B3).

[0015] Based on the above-mentioned kit, the present invention provides a method for isolating liver tissue cell nuclei. The cell nuclei are isolated using the above-mentioned kit, and the operation process is as follows: (1) Place the tissue sample to be processed in the lysis buffer LB Buffer, and lyse the cells by thorough grinding to obtain the initial lysis product. (2) Mix the initial lysis product with the washing and resuspension buffer WR Buffer to terminate the lysis, and obtain the crude cell nucleus extract through filtration, centrifugation, and resuspension. (3) Treat the crude cell nucleus extract with the debris removal components (B1 / B2 / B3) to separate the cell nuclei from the impurities. After washing the cell nuclei, high-quality cell nuclei are obtained.

[0016] For tissues of the same type, sometimes due to factors such as individual differences, preservation methods, preservation time, age, and diseases, the content of cell nuclei and impurities in the tissues vary greatly, and the optimal lysis time will also be different. The optimal lysis time should be adjusted according to the individual performance of the sample.

[0017] In the above-mentioned method, it is recommended that the usage amounts of the sample to be processed and the lysis buffer LB Buffer be 10 - 50 mg / 500 μL.

[0018] Immediately after lysis, mix the lysate with the pre-cooled washing and resuspension buffer WR Buffer. It is recommended to use 1 mL.

[0019] The entire experiment is carried out at 0 - 4 °C. Tissue lysis is carried out on ice, and the lysis time is 1 - 5 min.

[0020] Before tissue lysis: The reagents used are pre-cooled on ice for 10 min in advance. Aliquot 500 μL of the lysis buffer LB Buffer into a 1.5 mL EP tube, place the tissue in the lysis buffer, and quickly and thoroughly grind it 15 times with a grinding rod, and then incubate.

[0021] Among the methods of tissue fragmentation, compared with methods such as cutting with scissors, grinding has the characteristics of high lysis efficiency and good cell nucleus integrity, especially for samples with a small amount of tissue.

[0022] Use the debris removal components (B1 / B2 / B3) to separate the cell nuclei from the debris. Resuspend the cell nuclei with 400 μL of the washing and resuspension buffer WRBuffer, mix with 400 μL of the debris removal component B1, transfer to a pre-cooled 2 mL centrifuge tube, sequentially add 600 μL of the debris removal component B2 and 500 μL of the debris removal component B3 from the bottom of the tube, and centrifuge horizontally at 3000 g for 20 min at 4 °C, with the acceleration rate of 7 and the deceleration rate of 4.

[0023] In the above-mentioned method for isolating cell nuclei, a 40 μm filter membrane is used for filtration. If the finally obtained cell nuclei are aggregated, a 30 μm or smaller filter membrane can be used again for filtration.

[0024] Using this kit and the above separation method, even when the tissue amount is small, a sufficient amount of high-quality nuclear suspension can be obtained, which can be directly used for single-nucleus transcriptome sequencing without replacing the resuspension buffer.

[0025] The present invention provides a preferred solution, such as the extraction of liver tissue nuclei as described above, and the steps are as follows: (1) Before the experiment, add 1% BSA to the LB Buffer and use it immediately after preparation.

[0026] (2) Take 500 μL of LB Buffer added with 1% BSA and place it in a 1.5 mL EP tube.

[0027] (3) Quickly place a tissue block (fresh / frozen) the size of a pea (about 10 - 50 mg) and quickly grind the tissue using a grinding rod to make it homogenized (extrusion, rotation).

[0028] (4) Let it stand on ice for 1 - 5 min to fully lyse, and take a small amount of the suspension for microscopic examination every 30 s during this period until complete lysis.

[0029] (5) Quickly add 1 mL of WR Buffer and pipette and mix well on ice.

[0030] (6) Pass the suspension through a 40 μm cell strainer into a new 1.5 mL EP tube.

[0031] (7) Centrifuge at 500 G, 4 °C for 5 min.

[0032] (8) Remove the supernatant, being careful not to aspirate the precipitate, and add 400 μL of WR Buffer to resuspend the precipitate.

[0033] (9) Add 400 μL of B1, resuspend and mix well, and transfer the suspension to a new 2 mL EP tube.

[0034] (10) Slowly add 600 μL of B2 from the bottom of the tube to make the liquid layer.

[0035] (11) Then slowly add 500 μL of B3 from the bottom of the tube to make the liquid layer.

[0036] (12) Centrifuge horizontally at 3000 g, 4 °C for 20 min, with an acceleration rate of 7 and a deceleration rate of 4.

[0037] (13) After centrifugation, the nuclear layer is located at the interface between B2 and B3, and most of the impurities are located at B1 - B2. Remove the upper 800 μL of supernatant and impurities, and then remove the upper 200 μL.

[0038] (14) Aspirate the nuclear layer (white film layer) into 1 mL of WR Buffer and pipette and mix well.

[0039] (15) Centrifuge at 500 G for 5 min at 4 °C.

[0040] (16) Remove the supernatant, being careful not to aspirate the pellet, and resuspend the pellet in 500 μL of WR Buffer.

[0041] (17) Centrifuge at 500 G for 5 min at 4 °C.

[0042] (18) Remove the supernatant, being careful not to aspirate the pellet, and resuspend the pellet in 100 μL of WR Buffer.

[0043] (19) Take 5 μL of the nuclear suspension, stain it with trypan blue solution and examine it under a microscope to observe the quality of the cell nuclei; take 9 μL of the nuclear suspension, stain it with 1 μL of AO / PI dye, and count the cells using a Luna fl (fluorescence) cell counter.

[0044] The present invention provides a method for isolating cell nuclei from animal tissues, and the obtained cell nuclei can be directly used for single-cell nucleus transcriptome sequencing.

[0045] The quality control standards for the cell nuclei extracted by the present invention in single-cell nucleus transcriptome sequencing are as follows: the concentration is 500 - 1200 cells / μL, the cell viability is less than 5%, the aggregation rate is less than 5%, the volume is greater than 50 μL, most of the cell nuclear membranes are intact (greater than 90%), the cell nuclei have no obvious deformation, no rupture, no obvious debris and impurities.

[0046] The above-mentioned cell nucleus quality control standards can be detected by staining with 0.4% trypan blue, adding to a hemocytometer, and detecting the integrity of the cell nuclei under a microscope; fluorescence counting is performed using a Luna fl cell counter.

[0047] The present invention has the following advantages: (1) The tissue cell nucleus isolation kit provided by the present invention includes a cell nucleus lysis solution LB Buffer, a washing and resuspension solution WR Buffer, and a debris removal component (B1 / B2 / B3): Using WR Buffer for cell nucleus washing and resuspension can increase the stability of the cell nuclei and reduce the rupture of the cell nuclei.

[0048] LB Buffer has high lysis efficiency, can fully lyse the tissue within 1 - 5 min, generates less impurities, and at the same time has less impact on the cell nuclei and prevents RNA degradation.

[0049] The cell nuclei resuspended in WR Buffer can be directly used for single-cell nucleus transcriptome sequencing, simplifying the experimental operation and reducing the loss of cell nuclei.

[0050] (2)When extracting cell nuclei from tissues in the present invention, only a small amount of tissue (10 - 50 mg) is required.

[0051] (3)The debris removal components (B1 / B2 / B3) of the present invention simplify the removal of impurities in the cell nucleus suspension, can effectively remove impurities, and a high-quality single cell nucleus suspension can be obtained without the need for flow cytometry sorting.

[0052] (4)The tissue cell nucleus isolation kit and method provided by the present invention do not require expensive instruments such as flow cytometers, have low experimental costs, simple operations, and short experimental times (about 90 min), and can complete cell nucleus isolation and debris removal to obtain high-quality cell nuclei.

[0053] (5)The present invention has established higher cell nucleus quality control standards for single cell nucleus transcriptome sequencing. Implementing this quality control standard can obtain high-quality transcriptome data, and the cell nuclei extracted using this kit meet this quality control standard. Description of the Drawings

[0054] Figure 1 This is the quality control result of the mouse liver cell nuclei under the microscope in Case 1 of the present invention.

[0055] Figure 2 This is the quality control result of the mouse lung cell nuclei under the microscope in Case 2 of the present invention.

[0056] Figure 3 This is the quality control result of the mouse kidney cell nuclei under the microscope in Case 3 of the present invention.

[0057] Figure 4 This is the quality control result of the mouse brain cell nuclei under the microscope in Case 4 of the present invention. Detailed Embodiments

[0058] The following cases are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0059] The main reagents and consumables used in the following cases are as follows: Nonidet P40 Substitute is purchased from VWR, product number E109 - 100 mL; RNase inhibitor is purchased from Thermo Fisher Scientific, product number EO0384; DTT is purchased from Sigma, product number 646563; 40μm Cell Strainers are purchased from BIOFIL, product number CSS-013-040; OptiPrep™ Density Gradient Medium is purchased from Sigma, product number D1556; Trypan Blue Staining Solution (0.4%) is purchased from gibco, product number 15250-061; D-Hank's buffer is purchased from Procell, product number PB180321.

[0060] For Cases 1, 2, 3, and 4, fresh mouse liver tissue, lung tissue, kidney tissue, and brain tissue were used as materials respectively to isolate tissue cell nuclei and purify the cell nuclei. The specific operations are as follows: (1)Take 10 - 50 mg of fresh mouse tissue, wash it 2 - 3 times with pre - cooled 1xPBS, wipe dry the surface moisture, and immediately transfer it to a 1.5 mL EP tube; add 500 μL of pre - cooled LB Buffer, and grind it repeatedly 15 times on ice with a disposable grinding rod. Immediately start timing and incubate on ice for 1 - 5 min, and check under the microscope every 30 s.

[0061] The formula of the lysis buffer LB Buffer is as follows: Using D - Hank's buffer as the solvent, and additionally containing: Nonidet P40 Substitute 0.05 - 1%, MgCl 2 ·6H 2 O 3 - 5 mM, sucrose 250 - 300 mM, BSA 1%, Tween - 20 0.1%, RNase inhibitor 0.2 - 1 U / μL, DTT 1 - 2 mM; (2)When the lysis is sufficient, immediately add 1 mL of pre - cooled WR Buffer and mix well to terminate the lysis, obtaining the lysis product; The formula of the washing and resuspension buffer WR Buffer is as follows: Using D - Hank's buffer as the solvent, and additionally containing: 1% BSA, 0.2 - 1 U / μL RNase inhibitor, 1 - 2 mM DTT.

[0062] (3)Filter the lysis product through a 40 - μm filter membrane into a new 1.5 mL EP tube.

[0063] (4)Centrifuge at 500 g horizontally at 4℃ for 5 min; (5)Discard the supernatant, and resuspend the cell nuclei with 1 mL of pre - cooled WR Buffer.

[0064] (6)Centrifuge at 500 g horizontally at 4℃ for 5 min; (7)Discard the supernatant, resuspend the cell nuclei with 400 μL of pre - cooled WR Buffer, add 400 μL of B1, resuspend and mix well, and transfer the suspension to a new 2 mL EP tube.

[0065] (8)Slowly add 600 μL of B2 from the bottom of the tube to make the liquid layer.

[0066] (9)Then slowly add 500 μL of B3 from the bottom of the tube to make the liquid layer.

[0067] (10)Centrifuge at 3000 g at 4℃ horizontally for 20 min, with an acceleration rate of 7 and a deceleration rate of 4.

[0068] (11)The nuclear layer is located at the interface between B2 and B3. Most impurities are located at B1 - B2. Remove the upper 800 μL of supernatant and impurities, and then remove the upper 200 μL.

[0069] (12)Aspirate the nuclear layer (the white film layer) into 1 mL of WR Buffer and pipette to mix evenly.

[0070] (13)Centrifuge at 500 G, 4 °C for 5 min.

[0071] (14)Remove the supernatant, being careful not to aspirate the precipitate, and add 500 μL of WR Buffer to resuspend the precipitate.

[0072] (15)Centrifuge at 500 G, 4 °C for 5 min.

[0073] (16)Remove the supernatant, being careful not to aspirate the precipitate, and add 100 μL of WR Buffer to resuspend the precipitate.

[0074] (17)Take 5 μL of the nuclear suspension, stain it with trypan blue solution and examine it under a microscope to observe the quality of the cell nuclei; take 9 μL of the nuclear suspension, stain it with 1 μL of AO / PI dye, and count the cells using a Luna fl (fluorescence) cell counter.

[0075] Perform a quality inspection on the integrity of the cell nuclei using a microscope ( Figure 1 ), and count using a Luna fl cell counter; the nuclear membrane is intact, without rupture, and without obvious impurities and debris.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A kit for separating liver tissue cell nuclei comprises: a lysis solution LB Buffer, a cleaning and resuspension solution WR Buffer and a debris removal component B1 / B2 / B3; the lysis solution LB Buffer uses D-Hank's buffer as a solvent, and further comprises: 0.1% Tween-20, 1% BSA, 0.05-1% Nonidet P40 Substitute, 250-300mM sucrose, 3-5mMMgCl2·6H2O, 0.2-1U / μL RNase inhibitor, and 1-2mM DTT; the cleaning and resuspension solution WR Buffer uses D-Hank's buffer as a solvent, and further comprises: 1% BSA, 0.2-1U / μL RNase inhibitor, and 1-2mM DTT; the debris removal components B1 / B2 / B3 are respectively: B1: 40%-50% iodixanol, B2: 29%-31% iodixanol, and B3: 32%~50% iodixanol.

2. The kit according to claim 1, characterized in that The cell nuclear lysis solution LB Buffer uses D-Hank's buffer as a solvent, and further comprises: 0.1% Tween-20, 1% BSA, 0.05~1% Nonidet P40 Substitute, 250~300mM sucrose, 3~5mM MgCl2·6H2O, 0.2~1U / μL RNase inhibitor, and 1~2mM DTT.

3. The kit according to claim 1, characterized in that The cell nucleus washing and resuspension solution WR Buffer uses D-Hank's buffer as a solvent, and further comprises: 1% BSA, 0.2~1U / μL RNase inhibitor, and 1~2mM DTT.

4. The kit according to claim 1, characterized in that The fragment removal components B1 / B2 / B3 are respectively: B1: 40%-50% iodixanol, B2: 27%-33% iodixanol, and B3: 35%-50% iodixanol.

5. A method for isolating liver tissue cell nuclei, characterized in that: The kit according to any one of claims 1 to 4 is used to separate liver tissue cell nuclei, comprising: using the lysis solution LB Buffer to treat the sample to be treated to obtain a lysis product; mixing the lysis product with the cell nucleus washing and resuspension solution WR Buffer to terminate the lysis reaction; filtering, centrifuging and resuspending to obtain a crude cell nucleus extract; and treating the crude cell nucleus extract with the debris removal component B1 / B2 / B3 to obtain a high-purity cell nucleus.

6. The method for isolating liver tissue cell nuclei according to claim 5, characterized in that: The lysis condition is to be carried out on ice (0-4°C) and the lysis time is 1-5 min.

7. The method for isolating liver tissue cell nuclei according to claim 5, characterized in that: In the lysis treatment, the recommended usage amount of the sample to be processed and the lysis solution LB Buffer is 10-50 mg / 500 uL.

8. The method for isolating liver tissue cell nuclei according to claim 5, characterized in that: Immediately after the lysis, use pre-cooled washing and resuspension buffer WR Buffer to mix with the lysate. It is recommended to use 1 mL.

9. The method for isolating liver tissue cell nuclei according to any one of claim 5, characterized in that: The cell nucleus was separated from the debris using the debris removal components B1 / B2 / B3, the cell nucleus was resuspended with 400 μL of the washing and resuspension buffer WR Buffer, and mixed with 400 μL of the debris removal tissue fraction B1, and transferred into a pre-cooled 2 mL centrifuge tube, 600 μL of the debris removal tissue fraction B2 and 500 μL of the debris removal tissue fraction B3 were added from the bottom of the tube in sequence, and the tube was centrifuged horizontally at 2000-3000 g for 10-20 min at 4°C, with an ascending speed of 7 and a descending speed of 4.

10. The method for isolating liver tissue cell nuclei according to any one of claim 5, characterized in that: In the cell nucleus separation method, a 40 μm filter membrane is used for filtration. If the cell nuclei finally obtained are clumped, a 30 μm filter membrane may be used for filtration again.

11. A single cell nuclear transcriptome sequencing method, characterized in that: The method for separating liver tissue nuclei according to any one of claims 5 to 10 is used to separate the nuclei in the sample to be sequenced. By using the tissue nuclei separation kit and the above separation method, even when the amount of tissue is small, a sufficient amount of high-quality nuclei suspension can be obtained, which can be directly used for single-cell nuclear transcriptome sequencing without replacing the resuspension solution, and single-cell nuclear transcriptome sequencing is performed on the nuclei.

12. The single cell nuclear transcriptome sequencing method according to claim 11, characterized in that: The cell nucleus concentration is 700-1200 / μL, the cell activity is less than 5%, the clumping rate is less than 5%, the fragmentation rate is less than 5%, the volume is not less than 100 μL, and more than 90% of the cell nuclear membranes are intact and unbroken, and there are no obvious impurities.

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