Preparation method and application of single cell suspension of cold water fish gill tissue

Abstract

The invention belongs to the technical field of cell suspension preparation. Aiming at the problem that the single cell suspension of cold-water fish gill tissue prepared by the existing method maintains high activity for a short time, a preparation method and application of cold-water fish gill tissue single cell suspension are provided. By using the method of perfusing PBS through the heart of cold-water fish, the excess red blood cells in the gill tissue are removed, the gill tissue is sheared into a homogenate, centrifuged, the supernatant is discarded, a collagenase II solution is added, and the tissue is continuously rotated 360 degrees at 12-18 degrees Celsius until the tissue is completely digested and dissociated, filtered with a cell sieve, centrifuged, and the supernatant is discarded to obtain the cold-water fish gill tissue single cell single cell suspension. The method of the present invention is more sufficient in enzymatic hydrolysis, the experiment is short in time, and the operation steps are simple. The obtained cells have complete morphology, high cell concentration, cell activity of more than 90%, and agglomeration rate of less than 10%. The cell stability is good, and it can be stored for about 6 hours under 4 degrees Celsius environmental conditions, which can fully meet the needs of library construction of various single-cell sequencing platforms.

Description

Technical Field

[0001] The invention belongs to the technical field of cell suspension preparation, and particularly relates to a preparation method and application of a single cell suspension of cold-water fish gill tissue. Background Art

[0002] Cold-water fish is a type of fish that has strict requirements for the water temperature conditions of its living environment. It cannot grow and develop normally if the water temperature is higher than 20℃, so it is called cold-water fish. Rainbow trout is an important economic fish farmed worldwide. It is also a typical cold-water fish with advantages such as convenient feeding and management and easy access to materials. As one of the model species for fish scientific research, rainbow trout has been widely used in fields such as genomics and genetics. In addition, the cell diameter of rainbow trout is large, which is easy to observe and operate under a microscope. It is also one of the most widely used fish in single-cell experiments. The gill is an important tissue organ of fish. It is in direct contact with the external water environment and is very sensitive to changes in environmental factors such as temperature, salinity, and alkalinity in the water. It is a key tissue organ for fish to carry out gas exchange, ion transport, ammonia nitrogen metabolism, osmotic regulation, and hormone level regulation. The reason why the gill can achieve so many functions is inseparable from the various cell types formed by its specialization. Studies have found that fish gills include many different cell types, such as flat cells, columnar cells, neuroepithelial cells, progenitor cells, neutrophils, dendritic cells, mucus cells, ionocytes, and accessory cells. However, most of the current research remains at the level of gill tissue organs, and the results mainly reflect the information of the dominant group of cells in the gill tissue or the sum of information of multiple cell types, ignoring the heterogeneity between cells, resulting in the concealment or even loss of key information specific to the gill tissue cell subpopulations. Therefore, in order to deeply explore the biological functions of fish gill tissue, it is necessary to break through the previous technical bottleneck and conduct a more detailed study of molecular characteristics at the single cell level.

[0003] Single-cell sequencing technology refers to a new technology that performs high-throughput sequencing analysis of the genome, transcriptome, epigenome, and proteome at the level of a single cell. It can not only reveal differences in cell populations and cell evolutionary relationships, but also analyze the genetic structure and gene expression variation of a single cell, reflecting the heterogeneity between cells, and plays an important role in the fields of tumors, immunity, development, microbiology, and neurology. The key to single-cell sequencing technology is high-quality tissue cell suspension.

[0004] At present, the methods for processing animal tissue into single cell suspension mainly include mechanical method (shearing, grinding, blowing, rubbing on a net, etc.), chemical reagent treatment method (EDTA, EGTA cationic chelating agent, etc.), enzymatic method (pancreatin, collagenase, elastase, hyaluronidase, lysozyme), and enzymatic grinding method. CN106929468A discloses the effect of preparing single cell suspension of fish kidney tissue using different treatment methods.

[0005] Comparison table of the preparation effects of single cell suspension of crucian carp kidney tissue

[0006]

[0007] Currently, the preparation method of single-cell suspension of fish and other animal tissues mostly adopts mechanical grinding and water bath enzymatic hydrolysis. However, the cold-water fish cell suspension prepared by this enzymatic hydrolysis method has low cell activity and poor cell stability. It can usually only maintain high activity within 1 hour. It has high requirements on the experimental operation technology level, which is not conducive to the normal development of subsequent single-cell experimental work and seriously restricts the in-depth study of cell heterogeneity. Summary of the invention

[0008] In view of the above-mentioned problem that when the existing single-cell suspension preparation method is used to prepare cold-water fish gill tissue, the time for cells to maintain high activity is short. The present invention provides a single-cell suspension preparation method for cold-water fish gill tissue. The prepared single-cell suspension of gill tissue has more complete cell morphology, significantly improved cell stability, higher cell concentration, activity of more than 90%, sustainable stability for 6 hours, agglomeration rate of less than 10%, lower background signal generated by the cell suspension, short experiment time, low cost, high operability, can fully meet the construction of a single-cell transcriptome sequencing library, can also be used for cell nucleus extraction experiments, and has high application value.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A method for preparing a single cell suspension of cold-water fish gill tissue comprises the following steps:

[0011] S1. Remove excess red blood cells from gill tissue by perfusing PBS through the heart of cold-water fish. When the gill tissue turns light pink or white, quickly peel it off with surgical scissors sterilized by high temperature and high pressure, soak and wash it with PBS solution and DMEM / F12 solution respectively, cut the gill tissue into a homogenate with surgical scissors, and collect it into a sterile centrifuge tube;

[0012] S2, centrifuging the tissue homogenate obtained in step S1, discarding the supernatant, adding collagenase II solution, rotating continuously 360° at 12-18°C, and inspecting under an optical microscope until the enzyme completely digests and dissociates the tissue;

[0013] S3, filtering the single cell homogenate obtained in step S2 through a cell sieve, and washing the cold water fish cells remaining on the cell sieve with DMEM / F12, filtering the obtained filtrate again through a new cell sieve, washing the cell sieve with DMEM / F12, and transferring the filtrate to a centrifuge tube;

[0014] S4, centrifuging the enzymatic hydrolysate obtained in step S3, discarding the supernatant, adding DMEM / F12 solution to resuspend, and mixing the cell pellet; repeating the above centrifugation-resuspending operation to obtain the cold-water fish gill tissue single cell suspension.

[0015] Preferably, in step S1, a PBS solution is continuously perfused into the venous sinus above the heart of the cold-water fish using a medical sterile syringe.

[0016] Preferably, in step S1, the DMEM / F12 solution contains L-glutamine and HEPES (4-hydroxyethylpiperazineethanesulfonic acid) in a mass ratio of 1:1.

[0017] Preferably, in step S1, the pH of the PBS solution is 7.2-7.4, 0.01M.

[0018] Preferably, in step S2, collagenase II solution is added to the precipitate after centrifugation of the cold-water fish gill tissue homogenate, and the mixture is rotated 360 degrees continuously at 18° C. to mix and enzymatically digest for 1.5 to 3 hours.

[0019] Preferably, in step S2, the centrifugation temperature is 18°C, the speed is set to 200-300 RCF, the centrifugation time is 10s, the concentration of collagenase II solution is 2mg / mL, and collagenase II needs to be filtered and sterilized through a 0.22μm sterile filter membrane at the outer flame of an alcohol lamp.

[0020] Preferably, in step S3, the diameters of the cell sieves used are 70 μm and 40 μm, respectively. Preferably, the cell sieve is cleaned twice after each sieving.

[0021] Preferably, in step S4, the centrifugation temperature is 4°C and the rotation speed is 200 RCF.

[0022] Preferably, in step S4, centrifugation is performed twice, with the first centrifugation time being 3 min and the second centrifugation time being 6 min.

[0023] Preferably, in step S1, 100 mg of gill tissue is cut and homogenized, in step S2, 2 mL of collagenase II is used, in step S3, 200 μL of DMEM / F12 solution is used for washing the cell sieve each time, and in step S4, 200 μL of DMEM / F12 solution is used for resuspending each time.

[0024] The obtained single cell suspension of cold-water fish gill tissue was stained with AO / PI dye solution to detect the activity, concentration and aggregation rate of the cells.

[0025] Preferably, the mixing ratio of the AO / PI staining solution to the cell suspension is 1:1, and the staining time is 30 seconds.

[0026] The method for preparing the cold-water fish gill tissue single-cell suspension of the present invention can be applied to constructing a cold-water fish gill tissue single-cell transcriptome library, and can be applied to extracting cell nuclei from ATAC experimental samples.

[0027] The application includes placing the prepared single-cell suspension of cold-water fish gill tissue on ice and completing library construction or cell nucleus extraction within 6 hours.

[0028] Beneficial effects:

[0029] The preparation method provided by the present invention can quickly prepare a single cell suspension of cold-water fish gill tissue by shearing the gill tissue of the cold-water fish, and then undergoing continuous rotary digestion, double-layer filtration and single centrifugation. Compared with the commonly used water bath method, the method of the present invention has more complete enzymatic hydrolysis and better effect, and the experimental time can be shortened to 2-3 hours. Compared with the water bath enzymatic hydrolysis method, the experimental operation process is reduced, no water bath pot is required, the cost is low, the operation steps are simple, and the experimental feasibility is high. The obtained cell morphology is more complete than other methods, the cell fragments generated during the experiment are less, the cell concentration can reach more than 1600 / μL, the cell activity is more than 90%, and the agglomeration rate is less than 10%. The cell stability is good, and it can be stored for about 6 hours under 4°C environmental conditions, which can fully meet the needs of library construction of various single-cell sequencing platforms. The present invention provides a new technical solution for digging deep biological mechanism research of fish gill tissue, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A graph showing the quality inspection results (AO / PI) of a single cell suspension of rainbow trout gill tissue provided in Embodiment 1 of the present invention;

[0031] Figure 2 A quality inspection image of cell nuclei extracted from a single cell suspension of rainbow trout gill tissue provided in Embodiment 1 of the present invention;

[0032] Figure 3 A single cell sequencing map of a single cell suspension of rainbow trout gill tissue provided in embodiment 1 of the present invention;

[0033] Figure 4 A single cell sequencing map of a single cell suspension of rainbow trout gill tissue provided in embodiment 1 of the present invention;

[0034] Figure 5 This is a graph showing the quality inspection results (AO / PI) of single cell suspension of rainbow trout gill tissue in Example 2;

[0035] Figure 6 This is a graph showing the quality inspection results (AO / PI) of single cell suspension of rainbow trout gill tissue in comparative example 1;

[0036] Figure 7 This is the quality inspection result (AO / PI) of single cell suspension of rainbow trout gill tissue in comparative example 2;

[0037] Figure 8 This is the quality inspection result (AO / PI) of single cell suspension of rainbow trout gill tissue in comparative example 3. DETAILED DESCRIPTION

[0038] In order to explain the technical solution of the present invention more clearly, the present invention is further explained below in conjunction with specific embodiments.

[0039] In the following examples and comparative examples, unless otherwise specified, DMEM / F12 was purchased from VivaCell, China (Cat. No.: C3130-0500); collagenase II was purchased from Sigma, USA (Cat. No.: 1148090; CAS: 9001-12-1); and TESCA buffer was purchased from Solarbio, China (Cat. No.: G0150).

[0040] Example 1

[0041] 1. Sample preparation

[0042] S1. Use a sterile syringe to inject 10 mL of pH 7.2-7.4 0.01 M sterile PBS above the heart of rainbow trout. Perfuse the heart for 1 min to wash out a large number of red blood cells in the gill tissue. When the gill tissue turns light pink or white, use high-temperature and high-pressure sterilized scissors and forceps to dissect out the rainbow trout gill tissue. After washing with sterile PBS, cut 100 mg and put it into a 5 mL sterile enzyme-free centrifuge tube. Add 2 mL of DMEM / F12 (1:1) containing L-glutamine and HEPES to the centrifuge tube, and use scissors to cut the gill tissue into a homogenate.

[0043] S2. Use a 5mL sterile pipette tip to transfer the gill tissue homogenate to a 2mL sterile enzyme-free centrifuge tube, centrifuge at 18°C ​​at 200-300RCF for 10s, discard the supernatant, add 2mL 2mg / mL collagenase II solution to the precipitate, clamp the centrifuge tube on a horizontal shaker, perform enzymatic digestion at 18°C, and continuously rotate and digest for 1.5h. Remove the centrifuge tube from the mixer and slowly turn it upside down 3 times to ensure that the tissue homogenate in the tube can evenly contact the enzymatic mixture. Use a pipette to draw 12μL of cell suspension from the centrifuge tube, place it on a slide, cover it with a coverslip, and observe the state of cell dissociation under an optical microscope. If there are still tissue blocks that have not been completely digested into single cells in the cell suspension, extend the collagenase digestion time, and aspirate 12μL every 10 minutes for observation under a microscope until the tissue blocks are basically completely digested.

[0044] The preparation method of the collagenase II solution used is as follows: add 100 mg of collagenase II to 50 mL of TESCA buffer at pH 7.4, shake and mix until the collagenase II is completely dissolved, and filter with a 0.22 μm sterile filter membrane at the outer flame of an alcohol lamp to obtain a collagenase II mixed solution with a concentration of 2 mg / mL.

[0045] S3. Place a 70μm cell sieve on a 50mL sterile centrifuge tube, use a 5mL wide-mouth pipette tip to absorb the fully digested cell suspension, then slowly add it to the cell sieve with a 5mL pipette tip for filtration, add DMEM / F12 solution to wash the cell sieve twice, take out the filtered cell suspension, and slowly add it to a 40μm cell sieve for filtration again, and rinse the cell sieve twice with DMEM / F12 solution as a washing solution. After fully pipetting and mixing 10 times with a pipette, take out the second filtered cell suspension and transfer it to a 1.5mL centrifuge tube.

[0046] S4. Place the cell suspension prepared in step S3 into a centrifuge, set the temperature to 4°C, and the centrifugal speed to 200 RCF. After centrifugation for 3 minutes, a white precipitate can be observed at the bottom of the centrifuge tube. Use a large gun tip to absorb the supernatant on the upper layer of the precipitate, then add 200 μL of DMEM / F12 solution, and slowly blow and aspirate the precipitate 10 times with a large gun tip to evenly suspend the cells in the solution.

[0047] S5. Centrifuge the cell suspension in step S4 at 4°C and 200 RCF for 6 min, collect the white precipitate at the bottom of the centrifuge tube again in a 2 ml sterile centrifuge tube, and resuspend the precipitate with 200 μL DMEM / F12 solution. Quickly put the prepared cell suspension on ice to keep the temperature of the cell suspension at 4°C.

[0048] S6. Repeatedly pipette and mix the single cell suspension of gill tissue obtained in step S5, pipette 10 μL of the single cell suspension of gill tissue obtained in step S5, add 10 μL of AO / PI dye solution, mix thoroughly in a 1:1 ratio, pipette 20 μL of the mixture and add it to Countstar Rigel S3 for quality inspection, and measure cell activity, cell aggregation rate and cell concentration. The results are shown in Tables 1 and Figure 1 shown.

[0049] Table 1 Effect of single cell suspension preparation of rainbow trout gill tissue

[0050]

[0051] 2. Extraction of Cell Nuclei

[0052] The prepared single cell suspension was repeatedly pipetted and mixed, and 6×10 4 cells, the results of extracting cell nuclei are as follows Figure 2 shown.

[0053] like Figure 2 As shown, the cell nuclei of rainbow trout gill tissue are round or elliptical, the cell nuclei can be well separated, and the cell nuclei do not overlap each other. The results of the cell nuclei prove the integrity and efficiency of the technical solution of the present invention.

[0054] 3. Sequencing

[0055] The obtained single cell suspension of rainbow trout gill tissue was subjected to single cell RNA sequencing (scRNA-seq), and the results are as follows Figure 3 and Figure 4 shown.

[0056] like Figure 3 As shown in the figure, the cell clustering result diagram based on the total UMI number of each cell barcode, the horizontal and vertical coordinates of each point are obtained by the UMAP algorithm, each point represents a cell, and the color of each point is colored according to the UMI number, and the points of the same color are clustered into a cluster; compared with cells with low UMI numbers, cells with high UMI numbers may have high RNA content. Figure 4 As shown, a two-dimensional UMAP dimensionality reduction clustering diagram based on the color heat of the total number of UMIs per cell barcode is shown. Cells with similar gene expression profiles are clustered into a cluster. Each point represents a cell, and the colors of cells in different clusters are different.

[0057] like Figure 4As shown in the figure, the number of cells after sequencing is 6254, the median number of UMI per cell is 5015, the median number of genes per cell is 1297, and the average number of reads per cell is 108221; the turning point of the Beads to cells curve is obvious, indicating that the sequencing quality is very good and the interference background is small; Summary is the information of sample detection, the average expression abundance per cell is 9949, the total number of genes detected is 42491, the activity of gene expression per cell is 1753, and the number of cells used for clustering is 5634; Sequencing shows that the total number of sequencing reads is 1,830,571,803, and the reads after quality control for downstream analysis after filtering low-quality reads and invalid barcodes are 84.42%; M After filtering out low-quality reads and invalid barcodes in apping&Annotation, the number of quality-controlled reads used for downstream analysis was 1,545,341,473, with 94.89% of reads mapped to the genome, 49.03% of reads mapped to the positive strand, 45.86% of reads mapped to the antisense strand, 66.9% of reads mapped to the exon region, 3.7% of reads mapped to the intron region, and 8.2% of reads mapped to the antisense strand of the transcriptome.

[0058] Example 2

[0059] On the basis of Example 1, the enzymolysis temperature and time in step S2 are changed, and the specific operation is: the gill tissue homogenate is transferred to a 2mL sterile enzyme-free centrifuge tube with a 5mL sterile gun tip, and the rotation speed is 200-300RCF centrifuged for 10s at 18°C, and the supernatant is discarded. 2mL 2mg / mL collagenase II solution is added to the precipitation, and the centrifuge tube is clamped on a horizontal shaker, and the enzymolysis is performed at 12°C, and the continuous rotation digestion is performed for 3h. The centrifuge tube is removed from the mixer, and slowly inverted up and down 3 times to ensure that the tissue homogenate in the tube can be evenly contacted with the enzymolysis mixture. 12 μL cell suspension is drawn from the centrifuge tube with a pipette, placed on a slide, and after covering with a coverslip, the state of cell dissociation is observed under an optical microscope. If there are still tissue blocks that are not completely digested into single cells in the cell suspension, the digestion time of collagenase is extended, and 12 μL is sucked out and observed under a microscope every 10 minutes until the tissue blocks are substantially completely digested. Other operations are the same as in Example 1.

[0060] The cell activity, cell aggregation rate and cell concentration of rainbow trout gill tissue were measured. The results are shown in Table 2 and Figure 5 shown.

[0061] Table 2 Effect of single cell suspension preparation of rainbow trout gill tissue

[0062]

[0063] Comparative Example 1

[0064] On the basis of Example 1, change the enzymolysis temperature and time in step S2, specifically operate as follows: transfer the gill tissue homogenate to a 2mL sterile enzyme-free centrifuge tube with a 5mL sterile gun tip, centrifuge at room temperature speed 200-300RCF for 10s, discard the supernatant, add 2mL 2mg / mL collagenase II solution to the precipitation, place the centrifuge tube in a water bath, enzymolysis at 37°C, digest for 0.5h, take the centrifuge tube out of the water bath, slowly turn it upside down 3 times, ensure that the homogenate in the tube can evenly contact the enzymolysis mixture. Draw 12 μL cell suspension from the centrifuge tube with a pipette, place it on a slide, cover it with a coverslip, observe the state of cell dissociation under an optical microscope, if there are still tissue blocks that are not completely digested into single cells in the cell suspension, then extend the digestion time of collagenase II, every 10 minutes, suck out 12 μL and observe under a microscope until the tissue blocks are substantially all digested completely. Other operations are the same as in Example 1.

[0065] The activity, cell aggregation rate and cell concentration of the obtained rainbow trout gill tissue cells were measured. The results are shown in Table 3 and Figure 6 shown.

[0066] Table 3 Effect of single cell suspension preparation of rainbow trout gill tissue

[0067]

[0068] Comparative Example 2

[0069] Trypsin was purchased from Solarbio, China (Cat. No.: T1320).

[0070] On the basis of Example 1, the type of enzyme in step S2 is changed, and the specific operation is as follows: the gill tissue homogenate is transferred to a 2mL sterile enzyme-free centrifuge tube with a 5mL sterile gun tip, centrifuged at a speed of 200-300RCF for 10s at 18°C, the supernatant is discarded, 2mL 0.25% trypsin solution is added to the precipitate, the centrifuge tube is clamped on a horizontal shaker, 18°C ​​enzymolysis, continuous rotation digestion for 1.5h, the centrifuge tube is removed from the mixer, and slowly inverted up and down 3 times to ensure that the tissue homogenate in the tube can evenly contact the enzymolysis mixture. 12μL cell suspension is drawn from the centrifuge tube with a pipette, placed on a slide, and after covering with a coverslip, the state of cell dissociation is observed under an optical microscope. If there are still tissue blocks that are not completely digested into single cells in the cell suspension, the digestion time of trypsin is extended, and 12μL is sucked out every 10 minutes and observed under a microscope until the tissue blocks are basically completely digested. Other operations are the same as in Example 1.

[0071] The activity, cell aggregation rate and cell concentration of the obtained rainbow trout gill tissue cells were measured. The results are shown in Table 4 and Figure 7 shown.

[0072] Table 4 Effect of preparation of single cell suspension of rainbow trout gill tissue

[0073]

[0074] Comparative Example 3

[0075] On the basis of Example 1, the type of enzyme and the enzymolysis temperature in step S2 are changed, and the specific operation is: the gill tissue homogenate is transferred to a 2mL sterile enzyme-free centrifuge tube with a 5mL sterile gun tip, centrifuged at room temperature speed 200-300RCF for 10s, the supernatant is discarded, 2mL 0.25% trypsin solution is added to the precipitation, the centrifuge tube is placed in a water bath, 37°C enzymolysis, digestion 0.5h, the centrifuge tube is taken out from the water bath, slowly inverted up and down 3 times, and it is ensured that the tissue homogenate in the tube can be evenly contacted with the enzymolysis mixture. 12 μL cell suspension is drawn from the centrifuge tube with a pipette, placed on a slide, covered with a coverslip, and the state of cell dissociation is observed under an optical microscope. If there are still tissue blocks that are not completely digested into single cells in the cell suspension, the digestion time of trypsin is extended, and 12 μL is sucked out and observed under a microscope every 10 minutes until the tissue blocks are basically all digested completely. Other operations are the same as in Example 1.

[0076] The activity, cell aggregation rate and cell concentration of the obtained rainbow trout gill tissue cells were measured. The results are shown in Table 5 and Figure 8 shown.

[0077] Table 5 Effect of preparation of single cell suspension of rainbow trout gill tissue

[0078]

[0079] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a single cell suspension of cold water fish gill tissue, characterized in that: The steps include: S1. Remove excess red blood cells from gill tissue by perfusing PBS through the heart of cold-water fish. When the gill tissue turns light pink or white, quickly peel it off with surgical scissors sterilized by high temperature and high pressure, soak and wash it with PBS solution and DMEM / F12 solution respectively, cut the gill tissue into a homogenate with surgical scissors, and collect it into a sterile centrifuge tube; S2, centrifuging the tissue homogenate prepared in step S1 at 18°C, discarding the supernatant, adding collagenase II solution, rotating 360° continuously at 12-18°C for 1.5-3h, and inspecting under an optical microscope until the enzyme completely digests and dissociates the tissue; S3, filtering the single cell homogenate obtained in step S2 through a cell sieve, and washing the cold water fish cells remaining on the cell sieve with DMEM / F12, filtering the obtained filtrate again through a new cell sieve, washing the cell sieve with DMEM / F12, and transferring the filtrate to a centrifuge tube; S4, centrifuging the enzymatic hydrolysate obtained in step S3, discarding the supernatant, adding DMEM / F12 solution to resuspend, and mixing the cell pellet; repeating the above centrifugation-resuspending operation to obtain the cold-water fish gill tissue single cell suspension.

2. The preparation method according to claim 1, characterized in that: The cardiac perfusion method described in step S1 is: using a medical sterile syringe to slowly inject a sterile PBS solution into the venous sinus above the heart of the cold-water fish until the gill tissue turns light white.

3. The preparation method according to claim 1, characterized in that: The concentration of the collagenase II solution in step S2 is 2 mg / mL, and the collagenase II solution needs to be sterilized by filtering through a 0.22 μm sterile filter membrane.

4. The preparation method according to claim 1, characterized in that: The diameters of the cell sieves described in step S3 are 70 μm and 40 μm respectively.

5. The preparation method according to claim 1, characterized in that: The centrifugation temperature in step S4 is 4° C., the centrifugation speed is 200-300 RCF, and the centrifugation time is 3-6 min.

6. The preparation method according to claim 1, characterized in that: The cold water fish is rainbow trout.

7. Use of the method for preparing the cold-water fish gill tissue single-cell suspension according to claim 1 in constructing a cold-water fish gill tissue single-cell transcriptome library.

8. The use according to claim 7, characterized in that: The single cell suspension prepared according to claim 1 was placed on ice and the library was constructed within 6 hours.

9. Application of the method for preparing a single cell suspension of cold-water fish gill tissue according to claim 1 in extracting cell nuclei from ATAC experimental samples.

10. The use according to claim 9, characterized in that: The single cell suspension prepared according to claim 1 is placed on ice, and the cell nucleus is extracted within 6 hours.

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