Preparation method of single cell nucleus suspension of crustacean ovary

Abstract

The invention discloses a preparation method of a single cell nucleus suspension of a crustacean ovary, which comprises the following steps: cutting an ovary sample of a crustacean into pieces, grinding in a sterile enzyme-free centrifugal tube, adding a lysis solution for treatment for 5 minutes, immediately adding a washing solution for treatment, and filtering by using a filter membrane to obtain a filtrate; adding iodixanol into a centrifugal tube, then slowly spreading the filtrate above the iodixanol along the tube wall, then centrifuging and absorbing the supernatant to obtain a solution A, then slowly adding the resuspension into an original tube along the tube wall, centrifuging, and discarding the supernatant to obtain a cell nucleus layer A; taking another centrifuge tube, adding iodixanol, then slowly spreading the solution A above the iodixanol along the tube wall, then centrifuging, and discarding the supernatant to obtain a cell nucleus layer B; and combining the cell nucleus layer A and the cell nucleus layer B to obtain the single cell nucleus suspension to be detected. The method disclosed by the invention has the characteristics of mildness, rapidness and comprehensiveness, and is high in capability of capturing low-expression small cells, so that the detection of high-abundance and low-abundance cells is balanced.

Description

Technical Field

[0001] The invention belongs to the technical field of single cell nuclear suspension preparation, and in particular relates to a method for preparing a single cell nuclear suspension of crustacean ovaries. Background Art

[0002] The preparation of single-cell nuclear suspension is a key step in single-cell nuclear sequencing (scRNA-seq or snRNA-seq), and its quality directly affects the reliability of subsequent sequencing data and the interpretation of biological information. The preparation of single-cell nuclear suspension is the process of isolating single cell nuclei from tissue samples and suspending them in an appropriate solution, which mainly includes tissue fragmentation, filtration and washing, cell nucleus purification, resuspension and other steps. At present, people have established a set of mature preparation processes for mammalian cells, but due to the heterogeneity of crustacean tissues and cells, such mature processes may not be fully applicable. For example, the existing preparation technology has poor capture efficiency for low-abundance cell populations and poor ability to capture low-expressing small cells, which is not conducive to balancing the detection of high-abundance and low-abundance cells, making the obtained data unable to fully reflect the cellular heterogeneity of the sample. Summary of the invention

[0003] In view of the above shortcomings, the present invention discloses a method for preparing a single cell nuclear suspension of crustacean ovary, which is gentle, rapid and comprehensive, and has a strong ability to capture low-expressing small cells, thereby balancing the detection of high-abundance and low-abundance cells, improving the clarity of cell population analysis, and making the obtained data more comprehensive. Reflect the cellular heterogeneity of the sample.

[0004] The present invention is achieved by adopting the following technical solutions: A method for preparing a single cell nuclear suspension of crustacean ovaries, comprising the following steps: (1) A 10% NP-40 detergent solution, a 1M Tris-HCl buffer solution, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 40U / µL RNAse inhibitor solution and enzyme-free water are mixed to prepare a lysis solution; the volume ratio of the NP-40 detergent solution: Tris-HCl buffer solution: NaCl solution: CaCl2 solution: MgCl2 solution: RNAse inhibitor solution: enzyme-free water is 2:20:58.4:2:42:2:1874; (2) Prepare a washing solution by mixing 1 M Tris-HCl buffer, 5 M NaCl solution, 1 M CaCl2 solution, 1 M MgCl2 solution, 10% bovine serum albumin solution (BSA solution), 40 U / µL ribonuclease inhibitor solution (RNAse inhibitor) and enzyme-free water; (3) Prepare a resuspension by mixing PBS buffer, 10% bovine serum albumin solution (BSA solution) and 40 U / µL ribonuclease inhibitor solution; (4) Place a crustacean ovarian tissue sample in a sterile culture dish, cut the tissue sample into pieces with small surgical scissors, take out one-third of the sample and place it in a sterile enzyme-free centrifuge tube, then manually crush and grind it, add 1 to 3 mL of the lysis solution prepared in step (1) and treat it for 5 minutes, then immediately add an equal volume of the washing solution prepared in step (2) and treat it, and then filter it with a filter membrane to obtain a filtrate; (5) Add iodixanol to an enzyme-free centrifuge tube, then slowly spread the filtrate obtained in step (4) along the tube wall on top of the iodixanol to form two clear upper and lower layers of liquid, then centrifuge for 10 minutes, then aspirate the supernatant to obtain solution A, then slowly add the resuspension obtained in step (3) to the original tube along the tube wall and centrifuge for 10 minutes, discard the supernatant to obtain cell nuclear layer A; take another enzyme-free centrifuge tube and add iodixanol, then slowly spread the solution A along the tube wall on top of the iodixanol, then centrifuge for 10 minutes, discard the supernatant to obtain cell nuclear layer B; combine the cell nuclear layer A and the cell nuclear layer B to obtain the single cell nucleus suspension to be tested.

[0005] Furthermore, in step (2), the volume ratio of the Tris-HCl buffer:NaCl solution:CaCl2 solution:MgCl2 solution:bovine serum albumin solution:ribonuclease inhibitor solution:enzyme-free water is 100:292:10:210:1:10:9377.

[0006] Furthermore, in step (3), the volume ratio of the PBS buffer: bovine serum albumin solution: ribonuclease inhibitor solution is 8980:1000:20.

[0007] Furthermore, in step (4), a crustacean ovarian tissue sample is placed in a sterile culture dish, the tissue sample is chopped up with small surgical scissors, and one third of the sample is taken out and placed in a sterile enzyme-free centrifuge tube, followed by manual crushing and grinding for more than 10 times.

[0008] Furthermore, in step (4), the filter membrane is a 40 μm filter membrane.

[0009] Furthermore, in step (5), iodixanol is added to an enzyme-free centrifuge tube, and then the filtrate obtained in step (4) is slowly spread over the iodixanol along the tube wall to form two clear upper and lower layers of liquid, and then centrifuged at 1000×g for 10 minutes.

[0010] Furthermore, in step (5), the resuspension obtained in step (3) is slowly added to the original tube along the tube wall and centrifuged at 1000×g for 10 min, and the supernatant is discarded to obtain the cell nuclear layer A; the amount of the resuspension is 1.5 to 2 times the amount of the lysis solution.

[0011] Furthermore, in step (5), another enzyme-free centrifuge tube is taken and iodixanol is added, then the solution A is slowly spread on the iodixanol along the tube wall, and then centrifuged at 3000×g for 10 minutes, and the supernatant is discarded to obtain the cell nuclear layer B.

[0012] Compared with the prior art, this technical solution has the following beneficial effects: The present invention proposes a single cell nuclear suspension preparation technology suitable for complex crustacean tissues (such as heterogeneous tissues such as shrimp ovaries), which has excellent ability to capture low-expressing small cells, balances the detection of high-abundance and low-abundance cells, improves the clarity of cell population analysis, and the obtained data can more comprehensively reflect the cell heterogeneity of the sample. In addition, the method of the present invention is gentle, rapid, and comprehensive. Combined with HE staining sections, trypan blue staining, scanning electron microscopy, 10×Genomics quality control data, and t-SNE diagrams and other technical data verifications, it can be clearly shown that the method of the present invention significantly improves the capture efficiency and data reliability of low-abundance cell populations, and the obtained single cell nuclear suspension shows significant advantages in quality control indicators (such as FractionReads in Cells, Mitochondrial RNA) and cell clustering resolution (t-SNE diagram). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a picture of the ovary of the Penaeus vannamei dissected in Example 1.

[0014] Figure 2 This is a statistical result diagram of the ovarian index of three shrimps randomly selected in Example 1.

[0015] Figure 3 This is a panoramic scanning observation picture of HE-stained sections of the ovaries of three shrimps randomly selected in Example 1.

[0016] Figure 4 This is a flow chart of the method for preparing the single cell nucleus suspension described in Comparative Example 1.

[0017] Figure 5This is a flow chart of the method for preparing a single cell nucleus suspension as described in Example 1.

[0018] Figure 6 This is a photograph of the nuclear layer A and iodixanol obtained by centrifugation in the method for preparing a single cell nuclear suspension described in Example 1.

[0019] Figure 7 3 is a trypan blue staining microscopic examination picture of the cell nuclear suspension to be tested in the experimental example, wherein A is the cell nuclear suspension to be tested obtained by the method described in Comparative Example 1, and B is the cell nuclear suspension to be tested obtained by the method described in Example 1.

[0020] Figure 8 3 is a trypan blue staining microscopic examination picture of the supernatant of the test cell nuclear suspension after centrifugation in the experimental example, wherein A is the test cell nuclear suspension obtained by the method described in Comparative Example 1, and B is the test cell nuclear suspension obtained by the method described in Example 1.

[0021] Fig. 9 1 is a statistical result diagram of the cell nucleus recovery rate in the experimental example, wherein A is the cell nucleus suspension to be tested obtained by the method described in Comparative Example 1, and B is the cell nucleus suspension to be tested obtained by the method described in Example 1.

[0022] Fig.10 It is a scanning electron microscope image of the cell nuclei in the cell nucleus suspension to be tested obtained by the method described in Comparative Example 1 in the experimental example.

[0023] Fig.11 It is a scanning electron microscope image of the cell nuclei in the cell nucleus suspension to be tested obtained by the method described in Example 1 in the experimental example.

[0024] Fig.12 It is a t-SNE dimensionality reduction clustering diagram of a single cell transcriptome obtained by using the cell nucleus suspension to be tested obtained by the method described in Comparative Example 1 in the experimental example for single cell nucleus sequencing analysis.

[0025] Fig.13 It is a t-SNE dimensionality reduction clustering diagram of a single cell transcriptome obtained by using the cell nucleus suspension to be tested obtained by the method described in Example 1 in the experimental example for single cell nucleus sequencing analysis.

[0026] Fig.14 : is a quality control analysis graph of the cell nucleus suspension to be tested obtained by the method described in Comparative Example 1 in the experimental example. The first half of the curve represents the UMI distribution of real cells, and the second half gradually decreases to the background noise signal. The knee point of the curve is used to distinguish cells from the background.

[0027] Fig.15 : is a quality control analysis graph of the cell nucleus suspension to be tested obtained by the method described in Example 1 in the experimental example. The first half of the curve represents the UMI distribution of real cells, and the second half gradually decreases to the background noise signal. The knee point of the curve is used to distinguish cells from the background. DETAILED DESCRIPTION

[0028] The present invention is further described by the following examples, but is not intended to be limiting of the present invention. Specific experimental conditions and methods not specified in the following examples are conventional methods commonly known to those skilled in the art.

[0029] Example 1: Female Penaeus vannamei of 90 days old and 20.0 g ± 3.0 g were selected for the experiment. All experimental shrimps were provided by the Penaeus vannamei Genetic Breeding Center of Guangxi Academy of Fisheries Sciences.

[0030] First, three shrimps weighing 21 g ± 0.83 g were randomly selected, and their ovaries were carefully dissected after weighing the whole shrimps (see Figure 1 The ovary is semi-milky white and semi-transparent tissue. After dissection, there is no adhesion of other tissues. The weight of the ovary is recorded and the ovarian index (GSI) is calculated according to the following formula: Ovarian index = ovarian wet weight / total shrimp body weight × 100%. The results are shown in Figure 2 , the ovarian index was 0.182%±0.015%.

[0031] Then, the fresh ovary was flattened on filter paper, and the original morphology of the ovary was maintained before fixation in 4% paraformaldehyde for 24 hours; then, it was dehydrated under the following conditions: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65° for 1 hour, paraffin II melted at 65° for 1 hour, and paraffin III melted at 65° for 1 hour; the dehydrated tissue was embedded to obtain paraffin sections; after dewaxing the paraffin sections, they were placed in hematoxylin stain for 3-5 minutes, rinsed with tap water, and then placed in 95% alcohol for dehydration for 1 minute, and then placed in eosin stain for staining for 15 seconds, and finally dehydrated, sealed with neutral gum, and observed by panoramic scanning. The results are shown in the table. Figure 3 The nucleus size of the ovarian cells of the 20g white shrimp Litopenaeus vannamei ranged from 2.6µm to 24.3µm, all less than 40µm, meeting the requirements of the 10×Genomics platform.

[0032] Then, a single cell nuclear suspension of the ovary of Penaeus vannamei is prepared according to the method of the present invention, which comprises the following steps (see the process for details): Figure 5 ): (1) preparing a lysate by mixing a 10% NP-40 detergent solution, a 1M Tris-HCl buffer solution, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 40U / µL ribonuclease inhibitor solution and enzyme-free water; the volume ratio of the NP-40 detergent solution: Tris-HCl buffer solution: NaCl solution: CaCl2 solution: MgCl2 solution: ribonuclease inhibitor solution: enzyme-free water is 2:20:58.4:2:42:2:1874; (2) preparing a washing solution by mixing a 1M Tris-HCl buffer, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 10% bovine serum albumin solution, a 40U / µL ribonuclease inhibitor solution and enzyme-free water; the volume ratio of the Tris-HCl buffer: NaCl solution: CaCl2 solution: MgCl2 solution: bovine serum albumin solution: ribonuclease inhibitor solution: enzyme-free water is 100:292:10:210:1:10:9377; (3) preparing a resuspension by mixing a PBS buffer, a 10% bovine serum albumin solution, and a 40 U / µL ribonuclease inhibitor solution; the volume ratio of the PBS buffer: bovine serum albumin solution: ribonuclease inhibitor solution is 8980:1000:20; (4) Place the ovary of Penaeus vannamei in a sterile culture dish, cut the ovarian tissue 6 times with small surgical scissors to obtain a tissue sample, take out one-third of it and place it in a 5 mL enzyme-free centrifuge tube, then manually crush and grind it with a pestle for 10 times, then add 2 mL of the lysis solution prepared in step (1) and treat it for 5 minutes, then immediately add 2 mL of the washing solution prepared in step (2) to stop the lysis, and then filter it with a 40 μm filter membrane to obtain a filtrate; (5) Add 4 mL of iodixanol to a 15 mL enzyme-free centrifuge tube, then slowly spread the filtrate obtained in step (4) along the tube wall on top of the iodixanol to form two clear layers of liquid, then centrifuge at 1000 × g for 10 min, then carefully aspirate the supernatant to obtain solution A for later use, then slowly add 4 mL of the resuspension obtained in step (3) along the tube wall to the original tube and centrifuge at 1000 × g for 10 min, discard the supernatant and spread the remaining nuclear layer A on top of the iodixanol (see Figure 6 ); Take another new 15 mL enzyme-free centrifuge tube and add 4 mL iodixanol, then slowly spread the solution A on the iodixanol along the tube wall, and then centrifuge at 3000×g for 10 min, discard the supernatant, and spread the remaining cell nuclear layer B on the iodixanol; The nuclear layer A and the nuclear layer B are combined to obtain the single cell nucleus suspension to be tested.

[0033] Comparative Example 1: The ovaries of Penaeus vannamei were dissected according to the method described in Example 1, and then a single cell nucleus suspension was prepared according to the following steps (see the procedure for details). Figure 4 ): (1) A 10% NP-40 detergent solution, a 1M Tris-HCl buffer solution, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 40U / µL ribonuclease inhibitor solution and enzyme-free water were mixed to prepare a lysis solution; the volume ratio of the NP-40 detergent solution: Tris-HCl buffer solution: NaCl solution: CaCl2 solution: MgCl2 solution: ribonuclease inhibitor solution: enzyme-free water was 20:20:58.4:2:42:2:1856; the concentration of the NP-40 detergent in the lysis solution was much higher than that in the lysis solution described in Example 1; (2) preparing a washing solution by mixing a 1M Tris-HCl buffer, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 10% bovine serum albumin solution, a 40U / µL ribonuclease inhibitor solution and enzyme-free water; the volume ratio of the Tris-HCl buffer: NaCl solution: CaCl2 solution: MgCl2 solution: bovine serum albumin solution: ribonuclease inhibitor solution: enzyme-free water is 100:292:10:210:1:10:9377; (3) preparing a resuspension by mixing a PBS buffer, a 10% bovine serum albumin solution, and a 40 U / µL ribonuclease inhibitor solution; the volume ratio of the PBS buffer: bovine serum albumin solution: ribonuclease inhibitor solution is 8980:1000:20; (4) Place the ovary of Penaeus vannamei in a sterile culture dish, cut the ovarian tissue 6 times with small surgical scissors to obtain a tissue sample, take out one-third and place it in a 5 mL enzyme-free centrifuge tube, then manually crush and grind it with a pestle for 10 times, add 2 mL of the lysis solution prepared in step (1) and place it in a homogenizer for homogenization for 1 min, then immediately add 2 mL of the washing solution prepared in step (2) to stop the lysis, and then filter it with a 40 μm filter membrane to obtain a filtrate; centrifuge the filtrate at 500 × g for 5 min, discard the supernatant, then add 4 mL of the resuspension solution obtained in step (3) to resuspend the cell nuclei, centrifuge it at 500 × g for 5 min, discard the supernatant, and then add 1 mL of the resuspension solution obtained in step (3) to resuspend and obtain a single cell nucleus suspension to be tested.

[0034] Experimental Example: The quality of the single cell nucleus suspension prepared in Example 1 and Comparative Example 1 was evaluated, specifically including the following method: (1) Trypan blue staining to observe the morphology of the nucleus: Take 10 µL of the single cell nucleus suspension to be tested and mix it with 10 µL of trypan blue dye, then observe it under a microscope. The nucleus boundary must be clear and undamaged, and the nucleus suspension must not contain clumps larger than 40 µm or other granular impurities. The fragment rate in the suspension must be less than 20%, the clumping rate in the suspension must be less than 10%, and the total number of nuclei in the suspension must reach 10. 5 More than one; See the microscopic image Figure 7 and Figure 8 The nuclear membrane boundary of the cell nucleus suspension prepared by the method described in Comparative Example 1 is blurred, some cell nuclei appear non-circular in shape, there are many clumps and fragments, the homogeneity is poor, some cells are not completely lysed in the suspension, and some cell nuclei are lost in the supernatant, indicating that the conventional method described in Comparative Example 1 is not suitable for nuclear extraction from ovarian tissue of crustaceans; in the suspension prepared by the method described in Example 1, the cell nuclei are uniformly round with clear boundaries, the nucleolus structure inside the nucleus is consistent with the results of HE staining sections, the clumps and fragments are significantly reduced, and there is basically no loss of cell nuclei in the supernatant.

[0035] (2) Calculation of cell nucleus recovery rate: After each sample is lysed, the initial number of nuclei is recorded, and after resuspending with PBS RB and collecting the cell layer, the final number of nuclei is recorded, and the cell nucleus recovery rate is calculated. The calculation method is as follows: cell nucleus recovery rate = final number of nuclei / initial number of nuclei × 100%; when the method described in Comparative Example 1 is used, the initial number of nuclei is 4.6 million, and the final number of nuclei is 220,000; when the method described in Example 1 is used, the initial number of nuclei is 9 million, and the final number of nuclei is 3.65 million; the results of cell nucleus recovery rate are shown in Fig. 9 The cell nucleus recovery rate using the method described in Comparative Example 1 was only 4.93%, while the cell nucleus recovery rate using the method described in Example 1 was increased to 40.56%.

[0036] (3) Electron microscopy observation: Add 5 ml of electron microscopy fixative to 200 µL of single cell nucleus suspension, mix gently and immediately, fix at room temperature in the dark for 30 min, and store at 4°C. The fixed samples were post-fixed, dehydrated, dried, and treated with a conductive agent before observation under a scanning electron microscope. The results are shown in Fig.10 and Fig.11 ,Through electron microscopy observation, most of the cell nuclei in the suspension prepared by the method described in Comparative Example 1 were irregular in shape, with blurred outlines and damaged nuclear surfaces, while the cell nuclei in the suspension prepared by the method described in Example 1 were round in shape, with clear outlines, a reduced damage rate, and significantly reduced nuclear aggregation.

[0037] (4) Quality control evaluation of 10×Genomics platform: Single cell nuclear sequencing analysis was performed according to the official instructions of 10×Genomics Chromium Single Cell3' Library&Gel Bead Kit (Cat. No. 100007). The analysis results are shown in Figures 12 to 15 and Table 1.

[0038] Table 1 10×Genomics quality control report of single cell nuclear suspension

[0039] As shown in Table 1, compared with the method described in Comparative Example 1, the number of cells in the method of the present invention increased from 15,508 to 18,907, indicating that the method of the present invention improves the cell capture rate and better reflects the heterogeneity of the sample. Similarly, the distribution of the Clusters in the method of the present invention is more uniform, the internal structure of each group is more compact, and the number of cell subtypes is reduced (see Fig.12 and 13 ), indicating that the method of the present invention improves the homogeneity of gene expression within the population.

[0040] The Fraction Reads in Cells and Median UMI Counts per Cell of the method of the present invention have both decreased, but the total number of detected genes is basically the same, indicating that the comprehensiveness of gene coverage is not affected. At the same time, the subpopulation area in the t-SNE diagram has been reduced overall, and the distribution of small cell populations (such as Cluster 5 and Cluster 6) has become more concentrated and clear, which shows that the process scheme described in the present invention actually enhances the capture of low-abundance small cell subpopulations, improves the cell heterogeneity and comprehensiveness of the data, and reduces data bias.

[0041] In addition, the t-SNE diagram of the method of the present invention shows that the boundaries between clusters are clearer and the cell distribution is more compact, which indicates that while the number of reads decreases, the scheme of the present invention reduces the interference of non-target cells or noise signals.

[0042] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a single cell nuclear suspension of crustacean ovaries, characterized in that: The following steps are involved: (1) preparing a lysate by mixing a 10% NP-40 detergent solution, a 1M Tris-HCl buffer solution, a 5M NaCl solution, a 1M CaCl2 solution, a 1M MgCl2 solution, a 40U / µL ribonuclease inhibitor solution and enzyme-free water; the volume ratio of the NP-40 detergent solution: Tris-HCl buffer solution: NaCl solution: CaCl2 solution: MgCl2 solution: ribonuclease inhibitor solution: enzyme-free water is 2:20:58.4:2:42:2:1874; (2) Prepare a washing solution by mixing 1 M Tris-HCl buffer, 5 M NaCl solution, 1 M CaCl2 solution, 1 M MgCl2 solution, 10% bovine serum albumin solution, 40 U / µL ribonuclease inhibitor solution and enzyme-free water; (3) preparing a resuspension by mixing PBS buffer, a 10% bovine serum albumin solution, and a 40 U / µL ribonuclease inhibitor solution; (4) Place a crustacean ovarian tissue sample in a sterile culture dish, cut the tissue sample into pieces with small surgical scissors, take out one-third of the sample and place it in a sterile enzyme-free centrifuge tube, then manually crush and grind it, add 1 to 3 mL of the lysis solution prepared in step (1) and treat it for 5 minutes, then immediately add an equal volume of the washing solution prepared in step (2) and treat it, and then filter it with a filter membrane to obtain a filtrate; (5) Add iodixanol to an enzyme-free centrifuge tube, then slowly spread the filtrate obtained in step (4) along the tube wall on top of the iodixanol to form two clear upper and lower layers of liquid, then centrifuge for 10 minutes, then aspirate the supernatant to obtain solution A, then slowly add the resuspension obtained in step (3) to the original tube along the tube wall and centrifuge for 10 minutes, discard the supernatant to obtain cell nuclear layer A; take another enzyme-free centrifuge tube and add iodixanol, then slowly spread the solution A along the tube wall on top of the iodixanol, then centrifuge for 10 minutes, discard the supernatant to obtain cell nuclear layer B; combine the cell nuclear layer A and the cell nuclear layer B to obtain the single cell nucleus suspension to be tested.

2. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (2), the volume ratio of the Tris-HCl buffer:NaCl solution:CaCl2 solution:MgCl2 solution:bovine serum albumin solution:ribonuclease inhibitor solution:enzyme-free water is 100:292:10:210:1:10:9377.

3. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (3), the volume ratio of the PBS buffer solution: bovine serum albumin solution: ribonuclease inhibitor solution is 8980:1000:

20.

4. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (4), a crustacean ovarian tissue sample is placed in a sterile culture dish, the tissue sample is chopped with small surgical scissors, and one-third of the sample is placed in a sterile enzyme-free centrifuge tube, followed by manual crushing and grinding for more than 10 times.

5. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (4), the filter membrane is a 40 μm filter membrane.

6. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (5), iodixanol is added to an enzyme-free centrifuge tube, and then the filtrate obtained in step (4) is slowly spread over the iodixanol along the tube wall to form two clear upper and lower layers of liquid, and then centrifuged at 1000×g for 10 minutes.

7. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (5), the resuspension obtained in step (3) is slowly added to the original tube along the tube wall and centrifuged at 1000×g for 10 min. The supernatant is discarded to obtain the nuclear layer A; the amount of the resuspension is 1.5 to 2 times the amount of the lysis solution.

8. The method for preparing a single cell nuclear suspension of crustacean ovary according to claim 1, characterized in that: In step (5), another enzyme-free centrifuge tube is taken and iodixanol is added, then the solution A is slowly spread on the iodixanol along the tube wall, and then centrifuged at 3000×g for 10 minutes, and the supernatant is discarded to obtain the cell nuclear layer B.

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