Universal method for preparing animal tissue mononuclear suspension

Abstract

The invention provides a universal method for preparing animal tissue mononuclear suspension. A universal solution is provided for preparation of animal tissue single cell nucleuses, the method is suitable for different tissue types of various species, and the problem of unstable extraction caused by tissue differences in a traditional method is solved. Specifically, through multi-stage grinding and three dynamic quality inspection feedback mechanisms, hardness and cracking difficulty of different tissues are adapted, redundant operation is reduced to avoid damage of nuclear membranes caused by excessive grinding or excessive purification, nuclear membrane integrity and RNA stability are ensured, and extracted cell nucleuses are good in state and relatively high in integrity and can meet the requirement of single cell sequencing.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to a method for preparing single-cell nuclear suspensions, and more particularly to a general method for preparing single-cell nuclear suspensions from animal tissues. Background Art

[0002] Since the advent of single-cell RNA sequencing (scRNA-seq) technology in 2009, it has become a core tool for analyzing cell heterogeneity, developmental trajectories, and disease mechanisms. Especially in tumor research, it can reveal the characteristics of cancer cell subsets and promote the development of precision medicine. However, its application is limited by the preparation of high-quality single-cell suspensions, which not only requires cells to maintain a high survival rate but also ensures that mRNA is not degraded and its integrity is maintained. However, the tissue dissociation process is easily interfered by factors such as sample type and environmental conditions, resulting in the hindrance of technology promotion.

[0003] As an alternative, single-nucleus RNA sequencing (snRNA-seq) overcomes the sample preparation problems of scRNA-seq by focusing on RNA in the cell nucleus. It is applicable to frozen tissues or difficult-to-dissociate samples (such as brain tissues), reduces dissociation-induced transcriptional biases, and significantly improves data reliability. Its core technology lies in the efficient separation of cell nuclei: by mechanically / chemically lysing the cell membrane (such as enzymatic digestion of plant cells), protecting the integrity of the nuclear membrane with buffer solutions (such as liver tissues), and purifying by density gradient centrifugation (such as blood samples) to ensure the integrity of the nuclear structure and RNA quality.

[0004] Although there are currently various strategies for extracting single-cell nuclei from animal tissues, there are still many challenges in practical applications. There are significant differences in cell structures and compositions among different species, and the characteristics of tissue cells in different parts of the same species are also different. The muscle tissue cells and nerve tissue cells of animals have great differences in morphology, structure, and cell-to-cell connection methods. This makes it difficult to rely on a set of general extraction protocols to complete the extraction of cell nuclei from all species and tissues in different parts. For different research objects, it often takes a lot of time and effort to explore and optimize experimental conditions, which undoubtedly increases the complexity and difficulty of research. Summary of the Invention

[0005] In order to solve the problem of poor universality in the existing methods for extracting single-cell nuclei from animal tissues, the present invention provides a general method for preparing single-cell nuclear suspensions from animal tissues that is applicable to various animal tissues. When extracting cell nuclei from tissue samples such as the heart, liver, and muscle of different species such as mice, humans, chickens, and sugar gliders, this method can extract cell nuclei in good condition and with extremely high integrity, meeting the requirements of single-cell nuclear transcriptome sequencing for nuclear membrane integrity and RNA protection, and achieving the conditions for single-cell sequencing to be loaded onto the machine.

[0006] The technical solution adopted by the present invention is as follows: a general method for preparing a single-cell nuclear suspension of animal tissues, comprising the following steps: S1. Place the tissue sample in a tissue grinder containing a cell lysate, and vertically grind the tissue sample with grinding rod A to obtain a tissue homogenate; S2. Conduct a first quality inspection on the tissue homogenate to evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than a preset cell nucleus number threshold, pass the first quality inspection and execute step S6; otherwise, execute step S3; S3. Filter the tissue homogenate, transfer it to a clean tissue grinder, and vertically grind the tissue homogenate with grinding rod B; wherein, the gap between grinding rod B and the side wall of the tissue grinder base is smaller than the gap between grinding rod A and the side wall of the tissue grinder base; S4. Conduct a second quality inspection on the tissue homogenate to evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than the preset cell nucleus number threshold, pass the second quality inspection and execute step S6; otherwise, execute step S5; S5. Continuously vertically grind the tissue homogenate with grinding rod B, or let the tissue homogenate stand; repeat steps S4 to S5 until the tissue homogenate passes the second quality inspection and execute step S6; S6. Filter the tissue homogenate, centrifuge, and collect the precipitate; S7. Resuspend the precipitate with a cell nucleus washing solution to obtain a cell nucleus resuspension I; S8. Conduct a third quality inspection on the cell nucleus resuspension I to evaluate the amount of impurities contained in the cell nucleus resuspension I. If the amount of impurities is less than a preset impurity amount threshold, pass the third quality inspection and execute step S10; otherwise, execute step S9; S9. Purify the cell nucleus resuspension I with a cell nucleus purification solution, centrifuge, collect the precipitate, and resuspend the precipitate with a cell nucleus washing solution to obtain a cell nucleus resuspension II; S10. Centrifuge the cell nucleus resuspension I or the cell nucleus resuspension II, collect the precipitate, and resuspend the precipitate with a buffer solution to obtain a single-cell nuclear suspension.

[0007] Reference Figure 1, in order to complete the extraction of cell nuclei from animal tissues of different species and different parts through a set of general extraction schemes, the present invention adds three quality inspection processes to the conventional cell nucleus extraction method, and adopts different downstream schemes according to the quality inspection results for different degrees of tissue cell lysis and nuclear states. During the cell lysis process (Steps S1 to S5), the present invention sets two quality inspections to check the degree of cell lysis, and selects different operation steps according to the number of cell nuclei in the tissue homogenate. In the first quality inspection, if there are a large number of tissue clumps in the tissue homogenate that have not been released from the tissue after the grinding rod A finishes grinding, and the number of cell nuclei does not reach the preset cell nucleus number threshold, the first quality inspection fails and the grinding rod B with a finer grinding degree is used to continue grinding to avoid over-grinding and damaging the nuclear membrane. After the grinding rod B finishes grinding, the second quality inspection is carried out on the tissue homogenate. If the cell nuclei are fully released and the number of cell nuclei exceeds the preset cell nucleus number threshold, the second quality inspection is passed and the subsequent filtration operation is performed; otherwise, continue grinding or let the tissue homogenate stand to extend the lysis time. After the tissue homogenate is filtered and centrifuged, the precipitate is resuspended with a cell nucleus washing solution to wash the cell nuclei, and the third quality inspection is carried out on the obtained cell nucleus resuspension I. Whether to perform the cell nucleus purification operation (Step S9) is determined by evaluating the number of impurities (such as cell debris) contained in the cell nucleus resuspension I, protecting the integrity of the cell nucleus while reducing redundant operations. In the third quality inspection, if the number of impurities in the cell nucleus resuspension I exceeds the preset impurity number threshold, the cell nucleus resuspension I is purified with a cell nucleus purification solution (such as a cell nucleus purification solution containing iodixanol) to remove impurities. During the nuclear treatment process, adding a purification step to remove debris can effectively remove the interference of impurities and reduce the physical damage caused by cell debris to the cell nucleus, thereby significantly improving the quality of the cell nucleus and making it more pure and stable. Generally speaking, the present invention solves the instability problem caused by tissue differences in the traditional method by introducing real-time quality inspection (such as the cell nucleus number threshold, impurity number threshold, and those skilled in the art can independently set the threshold according to factors such as sample type and sample quality) in the preparation of single cell nuclei, provides a general solution for the preparation of single cell nuclei of animal tissues, can be applied to various tissue types of different species, and the obtained cell nuclei have a good state and relatively high integrity, which can meet the needs of single cell sequencing.

[0008] Preferably, the gap between the grinding rod A and the side wall of the tissue grinder base is 0.10 - 0.15 mm.

[0009] Preferably, the gap between the grinding rod B and the side wall of the tissue grinder base is 0.05 - 0.07 mm.

[0010] Preferably, the gap between the grinding rod A and the side wall of the tissue grinder base is 0.12 mm, and the gap between the grinding rod B and the side wall of the tissue grinder base is 0.06 mm. The vertical grinding of the tissue sample or tissue homogenate by the grinding rod A and the grinding rod B is to protect the integrity of the cell nucleus as much as possible and avoid the leakage of information such as RNA in the nucleus caused by nuclear breakage.

[0011] Preferably, the cell nucleus washing solution includes: 17-18 mM Tris pH 8.0, 170-180 mM sucrose, 40-45 mM KCl, 8-10 mM MgCl 2 , 0.1 mM DTT, 1X protease inhibitor, 0.03-0.05 U / μL RNase inhibitor, 0.5%-1.5% BSA.

[0012] Preferably, the cell nucleus purification solution includes: 20%-60% iodixanol, and the balance is the cell nucleus purification buffer. The volume fraction of iodixanol in the cell purification solution can be 20%, 30%, 40%, 50%, 60%. Those skilled in the art can determine the volume fraction of iodixanol in the cell purification solution according to the size of the cell nucleus in the sample, preferably 50%. If the diameter of the cell nucleus is very small (<5 μm), the proportion of iodixanol can be appropriately reduced, and if the cell nucleus is very large (>20 μm), the proportion can be appropriately increased. It should be noted that the cell nucleus purification solution provided by the present invention is different from the conventional cell nucleus purification system for density gradient centrifugation. Since the previous operations have obtained a cell nucleus resuspension I with good quality and few impurities, the present invention can directly add the described cell nucleus purification solution to the cell nucleus resuspension I to achieve the effect of removing debris, without adding it to the bottom of the cell nucleus resuspension and then performing density gradient centrifugation to separate and purify single cell nuclei as in the conventional method, thereby reducing redundant operations while protecting the integrity of the cell nucleus and saving operation time.

[0013] Preferably, the cell nucleus purification buffer includes: 18-22 mM Tris pH 7.8, 23-27 mM KCl, 3-7 mM MgCl 2 .

[0014] Preferably, in step S3, a 60-80 μm (preferably 70 μm) cell sieve is used to filter the tissue homogenate. Using a cell sieve with a larger pore size can filter out larger tissue blocks. If a conventional 40 μm cell sieve is selected, the tissue blocks may block the filter screen, resulting in a decrease in the yield of cell nuclei.

[0015] Preferably, step S3 further includes: cleaning the tissue grinder and the cell sieve with cell lysate, and collecting the cleaning solution into the tissue grinder with the filtered tissue homogenate. This step is carried out when the tissue homogenate obtained in step S1 is too little.

[0016] Preferably, in step S6, a 30-50 μm (preferably 40 μm) cell sieve is used to filter the tissue homogenate.

[0017] The present invention also provides an animal tissue single cell nucleus suspension obtained by treating with the method described above.

[0018] Advantages of the present invention: The present invention provides a general solution for the preparation of animal tissue single cell nuclei, which is applicable to different tissue types such as the heart, liver, and muscle of various species including mice, humans, chickens, sugar gliders, etc., and solves the problem of unstable extraction caused by tissue differences in traditional methods. Specifically, through multi-stage grinding (gradient design of the gap between grinding rods A / B) and a three-way dynamic quality inspection feedback mechanism (nucleus number threshold, impurity number threshold), it adapts to the hardness and lysis difficulty of different tissues, reduces redundant operations to avoid damage to the nuclear membrane due to excessive grinding or over-purification, ensures the integrity of the nuclear membrane and the stability of RNA, and the extracted cell nuclei are in a good state and have relatively high integrity, which can meet the requirements of single cell sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the experimental process of the present invention.

[0020] Figure 2 It is the first quality inspection diagram of Example 1 of the present invention; A is rat liver tissue, B is rat heart tissue, and C is rat stomach tissue.

[0021] Figure 3 It is the third quality inspection diagram of Example 1 of the present invention; A is before purification and B is after purification. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. In the embodiments of the present invention, unless otherwise specified, the methods used are all conventional methods, and the reagents used can be obtained from commercial channels.

[0023] Prepare the following solutions: The cell lysate includes: 17.4 mM Tris (Trishydroxymethylaminomethane) buffer pH 8.0, 173.8 mM sucrose, 43.5 mM KCl, 8.7 mM MgCl 2 , 0.1 mM DTT (1,4-Dithiothreitol), 1X protease inhibitor, 0.1 U / μL RNase inhibitor, 1% BSA (Bovine Serum Albumin), 0.05% Triton 100; among them, DTT, protease inhibitor, RNase inhibitor, BSA, and Triton 100 are added before use.

[0024] The nuclear washing solution includes: 17.7 mM Tris pH 8.0, 176.8 mM sucrose, 44.2 mM KCl, 8.8 mM MgCl 2 , 0.1 mM DTT, 1X protease inhibitor, 0.05 U / μL RNase inhibitor, 1% BSA.

[0025] The nuclear purification solution includes: 50% iodixanol, and the balance is nuclear purification buffer; the nuclear purification buffer includes: 20 mM Tris pH 7.8, 25 mM KCl, 5 mM MgCl 2 .

[0026] Example 1: In this example, single-cell nuclear suspensions were prepared using rat brain, rat liver, rat heart, mouse brain, and mouse kidney nuclei as samples. The specific steps are as follows.

[0027] S1. Insert the tissue grinder on ice and add 1.5 mL of pre-cooled cell lysate; at the same time, take a 1.5 mL centrifuge tube and insert it on ice, and add 500 μL of pre-cooled cell lysate. Take the tissue out of the liquid nitrogen / dry ice / -80°C refrigerator, take an appropriate size (for example, the size of a soybean, two mung beans, etc.) and place it in a 1.5 mL centrifuge tube containing cell lysate. Use surgical scissors to cut the tissue into 1 mm small pieces, and use a pipette tip to transfer the tissue pieces and liquid to the pre-cooled tissue grinder. Let it stand for 5 min until the tissue is fully infiltrated. Use grinding rod A (the gap between grinding rod A and the side wall of the tissue grinder base is 0.12 mm.) to vertically grind the tissue pieces until the resistance is significantly reduced (about 10 - 15 times) to obtain tissue homogenate.

[0028] S2. Conduct the first quality inspection on the tissue homogenate: Pipette 10 μL of the tissue homogenate, add 10 μL of AO / PI dye, place it in the countstar counter, observe whether the tissue is fully dissociated, and evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than the preset cell nucleus number threshold (the cell nucleus number threshold is generally set to 1000 nuclei / μL), then pass the first quality inspection and execute step S6; otherwise, execute step S3. S3. Filter the tissue homogenate through a 70 μm cell strainer and transfer it to a clean tissue grinder. If there is too little tissue homogenate, use 0.5 mL of cell lysate to wash the tissue grinder and the cell strainer, and collect the washing liquid into the tissue grinder containing the filtered tissue homogenate. Vertically grind the tissue homogenate 3 - 5 times with grinding rod B (the gap between grinding rod B and the side wall of the tissue grinder base is 0.06 mm) (do not rotate the grinding rod during the grinding process to avoid nuclear degradation).

[0029] S4. Conduct the second quality inspection on the tissue homogenate: Pipette 10 μL of the homogenate, add 10 μL of AO / PI dye, place it in the countstar counter, and evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than the preset cell nucleus number threshold, then pass the second quality inspection and execute step S6; otherwise, execute step S5.

[0030] S5. If the cell nuclei in the tissue mass are not fully released, then continue to vertically grind the tissue homogenate with grinding rod B. If the cell nuclei are separated individually but there is cytoplasm adhesion around them, let the tissue homogenate stand to extend the lysis time. Repeat steps S4 to S5 until the tissue homogenate passes the second quality inspection and execute step S6. S6. Filter the tissue homogenate through a 40 μm cell strainer, centrifuge at 4°C and 500 g for 5 minutes, and collect the precipitate. S7. Resuspend the precipitate with 1.5 mL of nuclear washing solution, gently pipette and mix well to obtain nuclear resuspension I. S8. Conduct the third quality inspection on nuclear resuspension I: Pipette 10 μL of the homogenate, add 10 μL of AO / PI dye, place it in the countstar counter, and evaluate the number of impurities contained in nuclear resuspension I. If the number of impurities is less than the preset impurity number threshold (the impurity number threshold is generally set to 20%), then pass the third quality inspection and execute step S10; otherwise, execute step S9. S9. Purify nuclear resuspension I with 1 mL of nuclear purification solution, centrifuge at 4°C and 800 g for 10 minutes, collect the precipitate, and resuspend the precipitate with nuclear washing solution to obtain nuclear resuspension II. S10. Centrifuge the nuclear resuspension solution I or nuclear resuspension solution II at 4 °C and 500 g for 5 minutes, collect the precipitate, and resuspend the precipitate with PBS buffer to obtain a single-cell nuclear suspension.

[0031] In the first quality inspection, different downstream protocols were adopted for the nuclear state after tissue grinding. See Figure 2 , use 10 μL of homogenate + 10 μL of AO / PI dye, and the red staining indicates the cell nucleus. Figure 2 In [reference figure], A is the rat liver tissue. There are still cell nuclei in the tissue that have not been released. Therefore, it is necessary to continue to perform step S3 and grind with the grinding rod B; Figure 2 In [reference figure], B is the rat heart tissue. There are still cell nuclei in the tissue that have not been released and the number of cell nuclei is small. Therefore, it is also necessary to continue to perform step S3 and grind with the grinding rod B; Figure 2 In [reference figure], C is the rat stomach tissue. The cell nuclei in the figure have been completely released and the number of cell nuclei exceeds the preset cell nucleus number threshold. Step S3 can be skipped and step S6 of filtration and centrifugation can be directly performed.

[0032] In the third quality inspection, observe the impurities in the nuclear resuspension solution I. See Figure 3 , Figure 3 In [reference figure], it is found that the unlabeled part in the background in A is the impurity. Then, use 1 mL of nuclear purification solution to purify the nuclear resuspension solution I. After purification, centrifuge and resuspend. At this time, it is found that the background of the obtained nuclear resuspension solution II is clean and the nuclear state is good ( Figure 3 in [reference figure] B), with high integrity. After centrifugation and resuspension with PBS, a single-cell nuclear suspension can be prepared, meeting the conditions for single-cell sequencing on the machine.

[0033] Perform single-cell nuclear transcriptome experiments on the single-cell nuclear suspension prepared from the above samples using MobiNova®-100, perform second-generation sequencing, and analyze the obtained sequencing results. The specific sequencing data analysis summary table is shown in Table 1. It can be seen that the nuclear extraction method provided by the present invention is applicable to various tissue types of different species, and the extracted single-cell nuclear suspension meets the requirements of single-cell sequencing with relatively high sequencing quality.

[0034] Table 1. Sequencing data analysis summary table Tissue type Rat brain Rat liver Rat heart Mouse brain Mouse kidney nucleus Initial tissue input <![CDATA[2 mung beans in size (55~65 mm 3 )]]> <![CDATA[1 soybean-sized (250 - 270 mm 3 ).]]> <![CDATA[2 soybeans in size (500 - 540 mm 3 ).]]> <![CDATA[1 soybean-sized (250 - 270 mm 3 ).]]> <![CDATA[1 soybean-sized (250~270 mm 3 ).]]> Nucleus aggregation rate 0.88% 1.34% 0.67% 1% 0.5% Total amount of nuclei 100w 167W 50w 80w 70w Number of valid reads 37,190,510 60,136,318 50,456,521 76,512,391 46,184,939 Proportion of valid reads 94.37% 92.31% 95.02% 92.02% 97.01% Genome-aligned reads 83.01% 95.59% 96.15% 93.44% 96.05% Estimated number of captured cells 3,117 3,496 5174 4,931 1,739 Median number of genes per cell 1,769 1,139 1207 1,458 1,814 Median number of UMIs per cell 3,751 1,927 2146 3,030 3,740 The above-described embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope of the present invention.

Claims

1. A general method for preparing a single cell nuclear suspension of animal tissue, characterized in that: The steps include: S1. The tissue sample is placed in a tissue grinder containing a cell lysis solution, and the tissue sample is vertically ground using a grinding rod A to obtain a tissue homogenate; S2. Performing a first quality inspection on the tissue homogenate to evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than a preset cell nucleus number threshold, the first quality inspection is passed and step S6 is executed, otherwise step S3 is executed; S3. Filter the tissue homogenate, transfer it to a clean tissue grinder, and vertically grind the tissue homogenate using a grinding rod B; wherein the gap between the grinding rod B and the side wall of the base of the tissue grinder is smaller than the gap between the grinding rod A and the side wall of the base of the tissue grinder; S4. Perform a second quality inspection on the tissue homogenate to evaluate the number of cell nuclei contained in the tissue homogenate. If the number of cell nuclei is not less than a preset cell nucleus number threshold, the second quality inspection is passed and step S6 is executed, otherwise step S5 is executed; S5. Continue to grind the tissue homogenate vertically using the grinding rod B, or allow the tissue homogenate to stand; repeat steps S4 to S5 until the tissue homogenate passes the second quality inspection and execute step S6; S6. Filter the tissue homogenate, centrifuge, and collect the precipitate; S7. Resuspending the precipitate using a cell nucleus washing solution to obtain a cell nucleus resuspension solution I; S8. Performing a third quality inspection on the cell nucleus resuspension solution I to evaluate the amount of impurities contained in the cell nucleus resuspension solution I. If the amount of impurities is less than a preset impurity amount threshold, the third quality inspection is passed and step S10 is executed, otherwise step S9 is executed; S9. Purifying the cell nucleus resuspension solution I using a cell nucleus purification solution, centrifuging, collecting the precipitate, and resuspending the precipitate using a cell nucleus washing solution to obtain a cell nucleus resuspension solution II; S10. Centrifuge the cell nucleus resuspension solution I or the cell nucleus resuspension solution II, collect the precipitate, and resuspend the precipitate with a buffer to obtain a single cell nucleus suspension.

2. The method according to claim 1, characterized in that The gap between the grinding rod A and the side wall of the tissue grinder base is 0.10-0.15 mm.

3. The method according to claim 1, characterized in that The gap between the grinding rod B and the side wall of the tissue grinder base is 0.05-0.07 mm.

4. The method according to claim 1, characterized in that The cell nucleus washing solution includes: 17~18 mM Tris pH8.0, 170~180 mM sucrose, 40~45 mM KCl, 8~10 mM MgCl2, 0.1 mM DTT, 1X protease inhibitor, 0.03~0.05 U / μL RNase inhibitor, and 0.5%~1.5% BSA.

5. The method according to claim 1, characterized in that The cell nucleus purification solution includes: 20% to 60% iodixanol, and the remainder is a cell nucleus purification buffer.

6. The method according to claim 5, characterized in that The cell nucleus purification buffer includes: 18~22 mM Tris pH 7.8, 23~27 mM KCl, 3~7 mM MgCl2.

7. The method according to claim 1, characterized in that In step S3, the tissue homogenate is filtered using a 60-80 μm cell sieve.

8. The method according to claim 7, characterized in that Step S3 also includes: using a cell lysis solution to clean the tissue grinder and the cell sieve, and collecting the cleaning solution into the tissue grinder with the filtered tissue homogenate.

9. The method according to claim 1, characterized in that In step S6, the tissue homogenate is filtered using a 30-50 μm cell sieve.

10. A single cell nuclear suspension of animal tissue obtained by processing using the method according to any one of claims 1 to 9.

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