Single cell nucleus extraction method for fresh cryopreserved tissue
Through wetting, grinding and lysis, filtration and multiple centrifugation treatments, combined with buffer without surfactant, the problems of low removal efficiency of myelin debris and large nuclear damage in single-cell extraction are solved, achieving high purity and high integrity nucleus extraction.
Patent Information
- Application Number
- CN202411329585.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-17
AI Technical Summary
During single-cell extraction, especially during brain tissue digestion, neuronal cells are prone to loss, and the myelin debris removal efficiency is low, resulting in low purity of the cell nucleus. The existing methods use surfactants to cause greater cell damage.
Using wetting, grinding and cleavage, Lysis Buffer and Wash Buffer without surfactant were used to remove myelin debris by diluted 50% Optiprep, reducing nuclear damage, improving purity and integrity .
Effectively remove myelin debris, reduce nucleus damage, improve nucleus extraction purity and integrity, reduce the probability of RNA degradation, and achieve efficient nucleus extraction.
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Figure CN119144697A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of cell nucleus extraction, and more specifically, to a single cell nucleus extraction method for fresh frozen tissue. Background Art
[0002] Since the birth of single-cell technology, it has been widely used in various research fields, such as neuroscience, tumor heterogeneity, cell differentiation, lineage tracing, treatment response, drug resistance, immune direction, and biomarker discovery.
[0003] Single-cell (or single-nucleus) sequencing technology, with its single-cell resolution and large-scale whole-transcriptome technology advantages, has unbiasedly presented the heterogeneity map of cell types within tissues, greatly promoting the development of various research fields. In addition, single-cell open chromatin group technology enables the molecular mechanism of transcriptome expression differences to be studied from an epigenetic perspective.
[0004] Since biology is extremely complex, scientists or researchers have been looking for new ways, methods and technologies to try to understand the mechanisms behind biology. Therefore, high-resolution technology can help researchers define or discover these heterogeneous cell populations, and based on these cell populations, discover new cell markers or related regulatory pathways, so as to compare the differences between healthy tissues and diseased tissues, find better solutions for the treatment of diseases, and make single cells effectively applied.
[0005] However, when preparing single-cell suspensions, most neuronal cells will be lost during the digestion of tissues such as the brain. At the same time, because the myelin sheath is smaller than the diameter of the cell, it cannot be removed by the sieve, which affects the detection of the cells.
[0006] In response to the above problems, the publication number is 2024103623192-A method for extracting mouse brain tissue nuclei based on high-density liquid technology, which is processed by lysis, filtration, centrifugation, resuspension and other processes, but the extraction auxiliary liquid used in this process usually adds a surfactant solution for auxiliary extraction. Due to the amphiphilicity of the surfactant, it can penetrate the cell membrane and enter the cell. Once entering the cell, it damages various structures within the cell, including the nucleus. Therefore, it is easy to cause the lysis time to be difficult to control, the damage to the nucleus is greater, and the efficiency of cleaning the myelin sheath fragments is low, and the purity of the obtained nucleus is also low. For this reason, further research is needed. Summary of the invention
[0007] In order to further improve the efficiency of myelin debris removal and reduce damage to cell nuclei, the present application provides a single cell nucleus extraction method for fresh frozen tissue.
[0008] The present application provides a method for extracting single-cell nuclei from fresh frozen tissue, comprising the following steps: 1) Mix the tissue with Lysis Buffer and let it stand to allow the tissue to be fully infiltrated to obtain a wetted substance A; 2) grinding and lysing the wetted material A to lyse the tissue and release the cell nuclei to obtain a lysate B; 3) The lysate B is blown and filtered, and the obtained filtrate is recorded as filtrate C; 4) Centrifuge the filtrate C, remove the supernatant, add Wash Buffer to the precipitate, pipette and resuspend, and obtain a resuspended product D; 5) Centrifuge the resuspended material D, remove the supernatant, add Diluted Nucleic Buffer to the precipitate, and resuspend it to obtain a resuspended material E; 6) Centrifuge the resuspended substance E, remove the supernatant, add Diluted Nucleic Buffer to the precipitate, resuspend, and pipette to obtain a cell nucleus suspension; Based on 5 mL Lysis Buffer, the Lysis Buffer comprises: 18-22 mM Tris, 24-26 mM KCL, 4-6 mM MgCL2, 240-260 mM Sucrose, 0.8-1.2% BSA, 0.5-1 U / μl RNase inhibitor, and is fixed to 5 mL with H2O; Based on 5 mL of Wash Buffer, the Wash Buffer comprises: 50% Optiprep with a mass concentration of 15-25%, 0.5-1 U / μl RNase inhibitor, and the volume is fixed to 5 mL with H2O; Based on 5 mL of 50% Optiprep, the 50% Optiprep contains: 60% Optiprep with a mass concentration of 48-52%, 18-22 mM Tris, 24-26 mM KCL, 4-6 mM MgCL2, and the volume is fixed to 5 mL with H2O.
[0009] In the above scheme, the present application removes other impurities in the tissue and improves the purity of the cell nucleus through wetting, grinding, lysis, filtration, and multiple centrifugation processes.
[0010] Neither the Lysis Buffer nor the Wash Buffer of the present application contains a surfactant, and therefore, the cell nucleus is not easily damaged during the extraction, lysis and washing processes. The 50% Optiprep used in the Wash Buffer of the present application is diluted by Basic Buffer, which can better separate impurities and cell nuclei without excessive stimulation to the cell nucleus, while 60% Optiprep will stimulate the cell nucleus, which is not conducive to the extraction of cell nuclei.
[0011] Among them, mM is the volume molar concentration (i.e. the amount of substance concentration), which refers to millimole per liter; U / μl is the concentration unit, and the unit U / μl represents the number of biologically active units (U) contained in each microliter (μl) of solution.
[0012] Tris is a buffer whose main component is tris(hydroxymethyl)aminomethane. The role of Tris is mainly reflected in maintaining the stability of RNA and DNA released after the sample to be extracted is lysed, and maintaining the pH value in the lysate. The preferred pH value of Tris in the present application is 7.8.
[0013] KCL and MgCL2 play the role of maintaining the osmotic pressure balance of cells and promoting the dissolution and extraction of proteins. Sucrose is sucrose, which acts as an osmotic pressure regulator to help maintain the osmotic pressure balance of cells, thereby promoting cell lysis. ABS is bovine serum albumin, which mainly protects the stability of enzymes and prevents enzyme decomposition and nonspecific adsorption. RNaseinhibitor is a ribonuclease inhibitor, which plays a role in reducing the activity of RNase released during cell disruption.
[0014] The Lysis Buffer obtained by 18-22mM Tris, 24-26mM KCL, 4-6mM MgCL2, 240-260mM Sucrose, 0.8-1.2% BSA, and 0.5-1U / μl RNase inhibitor has better lysis and penetration effects. Combined with the grinding and lysis process, the lysis effect of the tissue is improved, so that the efficiency of cell nucleus extraction can be improved.
[0015] 60% Optiprep is a 60% (w / v) iodixanol solution, which is further diluted with 18-22mM Tris, 24-26mM KCL, and 4-6mM MgCL2 to obtain Wash Buffer, which can better separate cell nuclei and myelin fragments in tissues, has less irritation to cell nuclei, and can easily separate cell nuclei from other impurities, thereby achieving better impurity removal, protecting cell nuclei to a greater extent, and improving the purity and integrity of cell nucleus extraction.
[0016] Lysis Buffer is Lysis Buffer; Wash Buffer is washing buffer; Diluted Nucleic Buffer is diluted nucleic acid buffer.
[0017] In summary, the wetting, grinding, lysis, filtration, and multiple centrifugal treatment processes of the present application, combined with the Lysis Buffer, Wash Buffer, etc. obtained in the present application, play a better role in removing impurities and separating cell nuclei. And the Lysis Buffer and Wash Buffer of the present application do not contain surfactants such as NP40 and Tween-20. Therefore, during the lysis process, the damage to the cell nucleus is small, and the preparation process of the present application can be combined to release the cell nucleus intact, and the probability of RNA degradation is greatly reduced; using the Wash Buffer containing Optiprep at a mass concentration of 15-25% 50%, combined with the centrifugal treatment process of the present application, the cell nucleus and impurities are separated, which is conducive to the efficient removal of impurities and incomplete cell nuclei, and improves the purity and integrity of cell nucleus extraction.
[0018] Preferably, based on 5 mL of Diluted Nucleic Buffer, the Diluted Nucleic Buffer comprises: BSA with a mass concentration of 0.8-1.2%, 0.8-1.2 U / μl RNase inhibitor, and the volume is fixed with H2O.
[0019] The diluent contains 0.8-1.2% BSA and 0.8-1.2U / μl RNase inhibitor, which can achieve a better dilution effect. Combined with the processes of wetting, grinding, lysis, filtration, and multiple centrifugation treatments, the Wash Buffer and Lysis Buffer configured in this application can remove myelin fragments in the tissue, reduce damage to the cell nucleus, and improve the extraction purity and integrity of the cell nucleus.
[0020] Preferably, the standing time in step 1) is 1.8-2.2 min.
[0021] The above standing time allows the Lysis Buffer to fully moisten the tissue, making it easier to grind the tissue into fine particles during subsequent grinding, facilitating tissue lysis and improving the extraction and removal efficiency of cell nuclei.
[0022] Preferably, in the step 2) of grinding and cracking, vertical grinding is performed for 15-20 times, and the mixture is allowed to stand for cracking for 1.5-2 minutes.
[0023] After 15-20 times of vertical grinding, the tissue can be ground into tiny particles, so that LysisBuffer can fully contact with the tiny particles, thereby promoting tissue lysis, allowing the cell nucleus to be completely separated, improving the extraction quality of the cell nucleus, and facilitating the removal of impurities such as myelin fragments, thereby improving the extraction purity of the cell nucleus.
[0024] Preferably, the blowing times in step 3) is 10-15 times, and the filtration mesh size is 30 μm.
[0025] By the above-mentioned blowing times, the raw material system of the resuspended material D is evenly dispersed, and by selecting a filter mesh with a pore size of 30 μm, impurities such as demyelinated debris can be removed to a greater extent, thereby improving the extraction purity and quality of the cell nucleus.
[0026] Preferably, the filter in step 3) needs to be rinsed with Lysis Buffer before filtration, and after filtration, the filter needs to be rinsed with Lysis Buffer 2-3 times.
[0027] By rinsing, impurities or residues on the filter can be removed to ensure that the filter will not be affected by these impurities when in use. At the same time, the activity of the filter can be maintained to ensure that the target substance can be effectively intercepted during formal filtration and cross contamination can be avoided. Rinsing the filter 2-3 times with Lysis Buffer can clean the cell nuclei remaining on the filter and improve the extraction efficiency of the cell nuclei.
[0028] Preferably, the centrifugation conditions of step 4) are 500g, 4σC, 5-6min, 9% up and 9% down.
[0029] The above uses a centrifugal force of 500g, centrifugation at 4°C, a centrifugation time of 5-6 minutes, and a centrifuge speed change mode of 9-9, that is, the centrifuge speed gradually increases to the maximum value (9) at the beginning, and then gradually decreases to the minimum value (9) at the end. The above conditions can help improve the separation effect, thereby achieving a better impurity removal effect.
[0030] Preferably, the centrifugation conditions of step 5) are 800g, 4°C, 5-10min, 4-up and 4-down centrifugation.
[0031] After the centrifugation treatment in step 4), the centrifugation conditions of 800g, 4°C, 5-10min, 4 steps up and 4 steps down are used again, and combined with the effect of Wash Buffer, a better centrifugation effect can be achieved, so that the cell nucleus and impurities can be effectively analyzed, so that the cell nucleus can achieve a better impurity removal effect and the integrity of the cell nucleus can be protected to a greater extent.
[0032] Preferably, the centrifugation conditions of step 6) are 500g, 4°C, 5-8min, 9% up and 9% down.
[0033] Further centrifugation is performed. The centrifugation conditions of 500g, 4°C, 5-8min, 9% up and 9% down can achieve a better centrifugal separation effect, so that the cell nucleus can achieve a better impurity removal effect.
[0034] Preferably, the tissue is one of brain, fat, placenta, spinal cord, and heart.
[0035] The single cell nucleus extraction method for fresh frozen tissue of the present application can be used for extracting cells from various tissues, such as extracting cell nuclei from brain, fat, placenta, spinal cord, heart and other tissues, and achieves a better extraction effect.
[0036] In summary, this application has the following beneficial effects: Through the wetting, grinding, lysis, filtration, and multiple centrifugation treatment processes of the present application, combined with the Lysis Buffer, Wash Buffer, etc. obtained in the present application, it plays a better role in removing impurities and separating cell nuclei. In addition, the Lysis Buffer and Wash Buffer of the present application do not contain surfactants such as NP40 and Tween-20. Therefore, during the lysis process, the damage to the cell nucleus is small, and the preparation process of the present application can be combined to release the cell nucleus intact, and the probability of RNA degradation is greatly reduced; using the Wash Buffer containing Optiprep with a mass concentration of 15-25% 50%, combined with the centrifugation treatment process of the present application, the cell nucleus and impurities are separated, which is conducive to the efficient removal of impurities and incomplete cell nuclei, and improves the purity and integrity of cell nucleus extraction. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 This is a diagram of the cell nucleus status of the mouse brain in Example 1.
[0038] Figure 2 This is a diagram of the nucleus status of white fat in mice in Example 2.
[0039] Figure 3 This is a diagram of the nuclear status of the mouse placenta in Example 3.
[0040] Figure 4 This is a diagram of the nuclear status of mouse mammary fat in Example 4.
[0041] Figure 5 This is a diagram of the nucleus status of the mouse spinal cord in Example 5.
[0042] Figure 6 This is a diagram showing the nuclear status of chicken liver in Example 6.
[0043] Figure 7 This is a diagram of the nuclear status of brown fat in mice in Example 7.
[0044] Figure 8 This is a diagram of the cell nucleus status of the mouse brain in comparative example 1. DETAILED DESCRIPTION
[0045] The following is combined with Figure 1-8 The present application is further described in detail with reference to the accompanying drawings and embodiments. Example
[0046] Example 1 A method for extracting single-cell nuclei from fresh frozen tissues, comprising the following steps: 1) Take 50 mg of tissue (fresh frozen mouse brain tissue) and put it into a 1.5 mL EP tube. Add 1 mL of Lysis Buffer to the EP tube containing the tissue, mix it, and let it stand for 2 minutes to allow the tissue to be fully infiltrated to obtain wetted material A; 2) Grind the wetted material A vertically for 15 times with a grinding rod, and do not rotate during the grinding process to prevent cell nucleus degradation. After grinding, let the tissue stand for 1.5 minutes to lyse, and obtain lysate B; 3) Rinse the 30 μm cell strainer thoroughly with 1 ml Lysis Buffer. At this time, the lysis time of lysate B reaches 2 min. Then, gently blow lysate B 10 times with a pipette, filter it again, and rinse the cell strainer twice with 2 ml Lysis Buffer. The filtrate obtained is recorded as filtrate C. 4) Centrifuge the filtrate C, remove the supernatant, add 5 mL of Wash Buffer to the precipitate, pipette 10 times, and resuspend to obtain a uniformly dispersed resuspended material D; 5) Centrifuge the resuspended material D, remove the supernatant, add 2 mL of Diluted Nucleic Buffer to the precipitate, and resuspend it to obtain a uniformly dispersed resuspended material E; 6) Centrifuge the resuspended material E, remove the supernatant, add 1 mL of Diluted Nucleic Buffer to the precipitate, resuspend, and pipette 10 times to obtain a uniformly dispersed cell nucleus suspension; Based on 5 mL of Lysis Buffer, Lysis Buffer contains: 20 mM Tris, 25 mM KCL, 5 mM MgCL2, 250 mM Sucrose, 1% BSA, 0.5 U / μl RNase inhibitor, and is fixed to 5 mL with H2O; Based on 5 mL of Wash Buffer, Wash Buffer contains: 50% Optiprep with a mass concentration of 20%, 0.5 U / μl RNase inhibitor, and the volume is adjusted to 5 mL with H2O; 50% Optiprep contains: 60% Optiprep with a mass concentration of 50%, 20mM Tris, 25mM KCL, 5mM MgCL2, and the volume is adjusted to 5mL with H2O.
[0047] Based on 5 mL of Diluted Nucleic Buffer, the Diluted Nucleic Buffer contains: 1% BSA by mass concentration, 1 U / μl RNase inhibitor, and is fixed to 5 mL with H2O.
[0048] The centrifugation conditions of step 4) are 500 g, 4° C., 5 min, 9% up and 9% down.
[0049] The centrifugation conditions of step 5) are 800 g, 4° C., 10 min, 4 up and 4 down centrifugation.
[0050] The centrifugation conditions of step 6) are 500 g, 4° C., 5 min, 9% up and 9% down.
[0051] Example 2 The difference between Example 2 and Example 1 is that the process conditions, the amount of raw materials used and the organization are different, as follows: The wetting time in step 1) is 1.8 min.
[0052] Step 2) During the grinding and cracking process, the product was vertically ground 18 times and allowed to stand for cracking for 1.4 minutes.
[0053] The number of blowing in step 3) is 12 times, and the mesh size of the filtering filter is 30 μm.
[0054] After filtration, the filter needs to be rinsed 3 times with Lysis Buffer.
[0055] The centrifugation conditions of step 4) are 500 g, 4° C., 8 min, 9 up and 9 down.
[0056] The centrifugation conditions of step 5) are 800 g, 4° C., 8 min, 4 up and 4 down centrifugation.
[0057] The centrifugation conditions of step 6) are 500 g, 4° C., 6 min, 9% up and 9% down.
[0058] Based on 5 mL Lysis Buffer, Lysis Buffer contains: 18 mM Tris, 24 mM KCL, 6 mM MgCL2, 240 mM Sucrose, 0.8% BSA, 0.8 U / μl RNase inhibitor, and is fixed to 5 mL with H2O; Based on 5 mL of Wash Buffer, Wash Buffer contains: 50% Optiprep with a mass concentration of 15%, 0.8 U / μl RNase inhibitor, and the volume is adjusted to 5 mL with H2O; Based on 5 mL of 50% Optiprep, 50% Optiprep contains: 60% Optiprep with a mass concentration of 48%, 18 mM Tris, 26 mM KCL, 6 mM MgCL2, and the volume is adjusted to 5 mL with H2O.
[0059] Based on 5mL Diluted Nucleic Buffer, Diluted Nucleic Buffer contains: 1.2% BSA, 0.8U / μl RNase inhibitor, and is diluted to 5mL with H2O.
[0060] The tissue is white adipose tissue of mice.
[0061] Example 3 The difference between Example 3 and Example 1 is that the process conditions, the amount of raw materials used and the organization are different, as follows: The wetting time in step 1) is 1.5 min.
[0062] Step 2) During the grinding and cracking process, vertical grinding was performed 20 times, and the mixture was allowed to stand for cracking for 1.2 minutes.
[0063] The blowing times in step 3) are 15 times, and the filtration mesh size is 30 μm.
[0064] After filtration, the filter needs to be rinsed 3 times with Lysis Buffer.
[0065] The centrifugation conditions of step 4) are 500 g, 4° C., 10 min, 9% up and 9% down.
[0066] The centrifugation conditions of step 5) are 800 g, 4° C., 5 min, 4 up and 4 down centrifugation.
[0067] The centrifugation conditions of step 6) are 500 g, 4° C., 8 min, 9 up and 9 down.
[0068] Based on 5 mL Lysis Buffer, Lysis Buffer contains: 22 mM Tris, 26 mM KCL, 4 mM MgCL2, 260 mM Sucrose, 1% BSA, 1 U / μl RNase inhibitor, and is fixed to 5 mL with H2O; Based on 5 mL of Wash Buffer, Wash Buffer contains: 50% Optiprep with a mass concentration of 25%, 0.5 U / μl RNase inhibitor, and the volume is adjusted to 5 mL with H2O; Based on 5 mL of 50% Optiprep, 50% Optiprep contains: 60% Optiprep with a mass concentration of 52%, 22 mM Tris, 24 mM KCL, 4 mM MgCL2, and the volume is adjusted to 5 mL with H2O.
[0069] Based on 5mL Diluted Nucleic Buffer, Diluted Nucleic Buffer contains: 0.8% BSA, 1.2U / μl RNase inhibitor, and is fixed to 5mL with H2O.
[0070] The tissue is mouse placenta tissue.
[0071] Embodiment 4-7 The difference between Examples 4-7 and Example 1 is that: the tissue sources are different, as shown in Table 1; Table 1 Tissue sources of Examples 1-7 Example Organization Source Example 1 Mouse brain tissue Example 2 White adipose tissue Example 3 Mouse placenta tissue Example 4 Mouse mammary adipose tissue Example 5 Mouse spinal cord tissue Example 6 Chicken liver tissue Example 7 Brown adipose tissue in mice Comparative Example Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the single cell nucleus extraction method for fresh frozen tissue comprises the following steps: 1) Prepare fresh frozen mouse brain tissue.
[0072] 2) Take 50 mg of tissue and put it into a 1.5 mL EP tube. First add 1 ml of Lysis Buffer and lyse for 30 seconds.
[0073] 3) After cutting the tissue with scissors, blow it 20 times with a pipette, fully lyse it for 2 minutes, and then filter it with a 30μm cell filter membrane.
[0074] 4) The filtrate obtained in step 3) was centrifuged at 500 g, 4° C., and 5 min. The centrifugation method was 9:9. The supernatant was removed, 2 ml of Wash Buffer was added to the precipitate, and then the precipitate was resuspended to obtain a first resuspension.
[0075] 5) Repeat step 4 twice to obtain a second heavy suspension.
[0076] 6) The second resuspension was centrifuged at 500 g, 4° C., 5 min, 4 up and 4 down, and the supernatant was removed. 3 ml of Diluted Nucleic Buffer was added to the precipitate, and the precipitate was resuspended to obtain the third resuspension.
[0077] 7) Centrifuge the second resuspension at 500g, 4°C, 5 min, 4 up and 4 down, remove the supernatant, add 1 ml of Diluted Nucleic Buffer to the precipitate, and resuspend to obtain a nuclear suspension.
[0078] Based on 5 mL of Lysis Buffer, the Lysis Buffer is divided as shown in Table 2; Lysis Buffer raw materials Based on 5 mL of Wash Buffer, the composition of Wash Buffer is shown in Table 3; Table 3 Raw materials of Wash Buffer Based on 5 mL of Diluted Nucleic Buffer, the composition of Diluted Nucleic Buffer is shown in Table 4; Table 4 Composition of Diluted Nucleic Buffer Performance testing Detection method / test method Use 0.4% trypan blue to count and hemocytometer to observe the cell nucleus status and count. Figure 1-8 .
[0079] Compare with Figure 1 and attached Figure 8 It can be seen that Figure 1 There are a small amount of impurities distributed in Figure 8There are dense impurities distributed in the solution, indicating that the preparation method and raw material dosage of the present application can achieve a better impurity removal effect to improve the extraction purity of the cell nucleus.
[0080] from Figure 2-7 It can be seen that Figure 2-7 The impurities are sparsely distributed and small in number, indicating that the preparation process of the present application can achieve better impurity removal effect on the extracted cell nuclei of different species and different tissues, thereby improving the extraction rate and purity of the cell nuclei.
[0081] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for extracting single-cell nuclei from fresh frozen tissue, characterized in that: The following steps are involved: 1) Mix the tissue with Lysis Buffer and let it stand to allow the tissue to be fully infiltrated to obtain wetted material A; 2) Grinding and lysing the wetted material A to lyse the tissue and release the cell nuclei to obtain lysate B; 3) The lysate B is blown and filtered, and the obtained filtrate is recorded as filtrate C; 4) Centrifuge the filtrate C, remove the supernatant, add Wash Buffer to the precipitate, pipette and resuspend to obtain resuspended material D; 5) Centrifuge the resuspended material D, remove the supernatant, add Diluted Nucleic Buffer to the precipitate, and resuspend it to obtain resuspended material E; 6) Centrifuge the resuspended material E, remove the supernatant, add Diluted Nucleic Buffer to the precipitate, resuspend, and pipette to obtain a cell nucleus suspension; Based on 5 mL of Lysis Buffer, the Lysis Buffer comprises: 18-22 mM Tris, 24-26 mM KCL, 4-6 mM MgCL2, 240-260 mM Sucrose, 0.8-1.2% BSA, 0.5-1 U / μl RNase inhibitor, and the volume is fixed to 5 mL with H2O; Based on 5 mL of Wash Buffer, the Wash Buffer comprises: 50% Optiprep with a mass concentration of 15-25%, 0.5-1 U / μl RNase inhibitor, and the volume is fixed to 5 mL with H2O; Based on 5 mL of 50% Optiprep, the 50% Optiprep contains: 60% Optiprep with a mass concentration of 48-52%, 18-22 mM Tris, 24-26 mM KCL, 4-6 mM MgCL2, and the volume is fixed to 5 mL with H2O.
2. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: Based on 5 mL of Diluted Nucleic Buffer, the Diluted Nucleic Buffer contains: BSA with a mass concentration of 0.8-1.2%, 0.8-1.2 U / μl RNase inhibitor, and the volume is fixed with H2O.
3. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The wetting time in step 1) is 1.8-2.2 minutes.
4. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: In the step 2) of grinding and cracking, vertical grinding is performed for 15-20 times, and the mixture is allowed to stand for cracking for 1.5-2 minutes.
5. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The blowing times in step 3) are 10-15 times, and the filtration mesh size is 30 μm.
6. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The filter in step 3) needs to be rinsed with Lysis Buffer before filtration, and after filtration, the filter needs to be rinsed with Lysis Buffer 2-3 times.
7. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The centrifugation conditions of step 4) are 500g, 4°C, 5-6 min, 9% up and 9% down.
8. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The centrifugation conditions of step 5) are 800 g, 4°C, 5-10 min, 4 up and 4 down centrifugation.
9. A method for extracting single cell nuclei from fresh frozen tissue according to claim 1, characterized in that: The centrifugation conditions of step 6) are 500g, 4°C, 5-8min, 9% up and 9% down.
10. A method for extracting single cell nuclei from fresh frozen tissue according to any one of claims 1 to 9, characterized in that: The tissue is one of brain, fat, placenta, spinal cord, and heart.