WO2022262102A1

WO2022262102A1 - Method for dissociation of bovine rumen epithelial tissue for single-cell sequencing

Info

Publication number: WO2022262102A1
Application number: PCT/CN2021/112671
Authority: WO
Inventors: 孙会增; 吴家劲; 刘潇菡; 朱森林; 顾凤飞; 刘建新
Original assignee: 浙江大学
Priority date: 2021-06-16
Filing date: 2021-08-16
Publication date: 2022-12-22
Also published as: CN113388570A; CN113388570B

Abstract

Provided is a method for the dissociation of bovine rumen epithelial tissue for single-cell sequencing, comprising: mechanical separation of rumen tissue, enzymatic digestion, filtration of impurities, cell washing, re-filtration, cell observation and quality control, dead cell removal, and the like. Said method can obtain a single-cell suspension of rumen tissue that has high activity, high cell count and low contamination, ensures the output of effective single-cell numbers and the quality of single-cell nucleic acid, and solves the problem that there is no systemic and perfect bovine rumen single cell dissociation technology in the existing technology.

Description

A method for dissociation of bovine rumen epithelial tissue for single-cell sequencing

technical field

The invention relates to the field of cell biology, in particular to a bovine rumen epithelial tissue dissociation method for single-cell sequencing.

Background technique

The rumen is a unique digestive organ of ruminants such as cows, which plays a vital role in the digestion, metabolism, absorption and transport of nutrients. Epithelial tissue is the main place where the rumen functions, but how to maintain and improve its structure and function in beef and dairy cattle farming still faces severe challenges, such as subacute rumen acidosis. Therefore, in-depth analysis of the key physiological functions and regulatory mechanisms of the rumen of dairy cows has become an important way to improve the health and production performance of dairy cows. Cells are the basic unit of life activities. The rumen epithelium is located on the innermost side of the rumen wall. It is a stratified squamous epithelium with four layers of multifunctional cell layer structure. The protruding layer, granular layer and stratum corneum, but the study of their cell types and molecular characteristics is still obviously insufficient.

At present, most studies on the function of dairy cow rumen epithelial tissue are at the tissue level or on rumen epithelial cells cultured in vitro. By analyzing the transcriptome of rumen epithelial tissue, we captured all the expressed genes under specific conditions, and then analyzed the changes in system function under different external treatments or stimuli. However, 1 gram of rumen epithelial tissue contains at least millions of cells. Transcriptome analysis at the traditional tissue level ignores cell heterogeneity and cannot obtain functional characteristics at the cell level. In the method based on the cell level, the purified cell suspension contains various types of cells such as basal layer, spinous process layer and even granular layer cells, so the existing research is based on a large class of epithelial cells and cannot be in-depth This also leads to the fact that the function of dairy cow rumen epithelial tissue cannot be accurately analyzed at the cellular level; at the same time, the interaction between different types of cells in rumen epithelial tissue, the function of a small number of mesenchymal cells, A series of basic and important issues such as the trajectory of cell differentiation and development.

An important prerequisite for single-cell level research is to establish a method for sufficient tissue dissociation and single-cell suspension preparation that meets the conditions for single-cell capture and tracking; the biggest difference compared with rumen epithelial cell isolation and culture methods is that this type of method requires Tissues are sufficiently dissociated to obtain all cell types, and these cells can be efficiently captured and specifically tracked. Our previous research also found that the cell suspension obtained by the traditional rumen epithelial cell isolation and culture method in vitro cannot meet the requirements of single-cell sequencing, and further exploration is needed in the method. The invention successfully establishes for the first time the technology of rumen epithelial tissue dissociation and single cell suspension preparation for single cell sequencing such as single cell transcriptome, which will promote the development of rumen single cell research in ruminants. At the same time, it will establish a good foundation for other single-cell-based research in the future, such as single-cell epigenetics and single-cell proteomics.

Contents of the invention

The purpose of the present invention is to address the deficiencies of the prior art, to propose a bovine rumen epithelial tissue dissociation method for single-cell sequencing, to obtain a single-cell suspension of rumen tissue with high activity, high cell number, and low pollution, and to ensure effective single-cell The output of data and the quality of single-cell nucleic acid are convenient for single-cell omics sequencing and data analysis.

The object of the present invention is achieved by the following technical solutions: a bovine rumen epithelial tissue dissociation method for single-cell sequencing, specifically comprising the following steps:

(1) Mechanical separation of rumen tissue

Put a sterile RNase-free cell culture dish on an ice box and add PBS; put the rumen tissue in the culture dish, wash it with DPBS, use sterilized surgical scissors to remove the muscle layer, cut the tissue pieces into pieces, and put them in Soak in EDTA, then wash with DPBS again, continue to cut the tissue, and discard the supernatant;

(2) Enzyme digestion treatment

Add trypsin/EDTA to the last cut tissue piece in step (1) for digestion; after digestion, insert into ice and add HBSS to stop digestion; after centrifugation, wash again with HBSS; add the incubated enzyme dissociation solution, and the enzyme dissociation The solution contains neutral protease, collagenase I, collagenase IV, hyaluronidase and DNase I, after incubation, centrifuge; then add FBS to inhibit enzyme activity, and obtain a digested cell suspension;

(3) Impurity filtration

Filter the digested cell suspension in step (2) with a cell strainer and collect it into a new EP tube; discard the supernatant after centrifugation;

(4) Cell washing

Add HBSS to the cell pellet after centrifugation in step (3) to resuspend, discard the supernatant after centrifugation; add PBS containing BSA for resuspension, discard the supernatant after centrifugation; add PBS containing BSA to resuspend the cells to obtain a cell suspension liquid;

(5) filter again

Use a cell strainer to filter the cell suspension obtained in step (4), centrifuge after washing the filter membrane with PBS containing BSA; observe the cell debris, if the debris rate is greater than 10%, repeat this step;

(6) Cell observation and quality inspection

Blow evenly the cell suspension obtained in step (5), take part of the cell suspension and place it in a hemocytometer, observe the background debris, cell clumping and cell density with a microscope; add trypan blue staining, calculate cell viability, cell concentration and total cells. If it is observed that the cell activity does not meet the requirements for cell activity of single-cell sequencing, but the total number of cells is sufficient, the dead cell removal operation is required.

Further, if it is observed in step (6) that the cell activity does not meet the cell activity requirements of single-cell sequencing, but the total amount of cells is sufficient, a dead cell removal device can be used to remove dead cells. The specific operation is as follows: In the ultra-clean bench, dilute 20×Binding Buffer with double distilled water to 1×Binding Buffer; centrifuge the cell suspension obtained in step (5) and discard the supernatant; collect the cell pellet, add Dead Cell Removal MicroBeads were blown and mixed; incubated at room temperature, during which the diluted Binding Buffer was added; after the incubation, before the washing solution exposed the column, the diluted Binding Buffer was added to the cell suspension and added to the top of the column; washed with the diluted Binding Buffer Column; centrifuge the collection tube and discard the supernatant; add BSA-containing PBS to resuspend the cells; mix by pipetting and continue to step (6) for observation.

Further, in step (1), the tissue block is first chopped into small pieces with a volume of 10×0.5 mm ² , and when the tissue is further chopped, the tissue block is chopped until the volume of the tissue block is 1 mm ³ . The PBS is 1×PBS, and the amount added is 5 ml; the DPBS is 1×DPBS. The ratio of the volume of EDTA added to the volume of the tissue block is 2:1.

Further, in step (1), the EDTA concentration is 20 mM, and soaked at 37° C. for 30 min.

Further, in step (2), the concentration of the trypsin/EDTA is 2500mg/L, and the ratio of the added volume to the tissue block after the tissue is further shredded is 2:1; the digestion conditions are: digestion temperature The temperature is 37°C, and the digestion time is 5 minutes. The cooling time in ice is 2 min. The volume of the HBSS is equal to that of the cell suspension, and the HBSS is pre-cooled HBSS; the centrifugation conditions are: the centrifugation speed is 300×g, the centrifugation temperature is 4° C., and the centrifugation time is 2 minutes. The ratio of the volume of the enzyme dissociation solution added to the tissue block after the tissue is continued to be shredded is 2:1, which contains 1.5mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I; the reaction conditions are: the incubation temperature is 37°C, and the incubation time is 30min. The FBS is 10% FBS (V/V), and the volume is the same as that of the enzyme dissociation solution added.

Further, in step (3), the filtration uses stacked cell filters with pore sizes of 30 μm and 70 μm. The centrifugation conditions are as follows: the centrifugation speed is 300×g, the centrifugation temperature is 4° C., and the centrifugation time is 5 minutes.

Further, in step (4), the amount of HBSS added is 2 mL. The amount of PBS added was 2 mL; the PBS was 1×PBS, and the PBS contained BSA at a concentration of 400 mg/L. The centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.

Further, in the step (5), the cell filter uses a Miltenyi 20 μm cell filter; the PBS is 1×PBS, and the PBS contains BSA at a concentration of 400 mg/L. The centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.

Further, the device for removing dead cells includes MS or LS columns, magnets, racks, and receiving tubes. For MS or LS, the amount of Binding Buffer used is different. The diluted Binding Buffer is 1×Binding Buffer; the usage condition of MS is 3-4ml/rxn, and the usage condition of LS is 17-26ml/rxn. The amount of Binding Buffer added during incubation is: add 500μl to MS, add 3ml to LS; after incubation, the amount of Binding Buffer added is: add 500-1000μl to MS; add 1-10ml to LS. When washing the column, the amount of Binding Buffer added is: 500μl each time in MS, 4 times in total; 3ml each time in LS, 4 times in total.

Further, the amount of Dead Cell Removal MicroBeads added is 100 μl. The incubation time is 15 minutes, and the time of adding Binding Buffer is the 14th minute of incubation; the centrifugation conditions are: the centrifugation speed is 300 × g, the centrifugation temperature is 4°C, and the centrifugation time is 5-10 minutes. The amount of PBS added is 50-300 μl; the PBS is 1×PBS, and the PBS contains BSA at a concentration of 400 mg/L.

Compared with the prior art, the beneficial effects of the present invention are as follows: there is no specific and systematic single-cell dissociation method for bovine rumen tissue, and the present invention achieves a new breakthrough. The invention can obtain single-cell suspension of rumen tissue with high activity, high cell number, low pollution and complete cell types, ensuring the output of effective single-cell number and the quality of single-cell mRNA, so as to facilitate single-cell sequencing and data analysis.

Description of drawings

Fig. 1 is the image under the microscope of bovine rumen tissue single-cell suspension that parallel experiment (1) obtains in the specific embodiment: background debris is less, cell clumping situation almost does not have, and cell density is moderate;

Fig. 2 is the image under the microscope of bovine rumen tissue single-cell suspension that parallel experiment (2) obtains in the specific embodiment: background debris is less, the cell clumping situation hardly has, and cell density is moderate;

Fig. 3 is the image under the microscope of bovine rumen tissue single-cell suspension obtained in parallel experiment (3) in the specific embodiment: background fragments are few, cell agglomeration situation almost does not have, and cell density is moderate;

Fig. 4 is the cell concentration of the bovine rumen tissue single cell suspension that parallel experiment (1) in the specific embodiment, (2) and (3) obtains, and sample one, two, three are used in Fig. 1,2,3 respectively The samples are the same: the cell concentrations are 1.15×10 ⁶ /mL, 1.27×10 ⁶ /mL and 2.64×10 ⁶ /mL, exceeding 10 ⁶ /mL;

Fig. 5 is the cell viability rate of the bovine rumen tissue single cell suspension that parallel experiment (1) in the specific embodiment, (2) and (3) obtains, and sample one, two, three are respectively with Fig. 1,2,3. The same sample was used: the cell viability was 88%, 94% and 94% respectively, reaching the standard of 85%;

Fig. 6 is the cDNA quality inspection figure of the bovine rumen tissue single-cell suspension obtained in the specific embodiment: the main peak of the fragment distribution figure is greater than 1000bp and there is no jagged miscellaneous peak, and it is judged as qualified;

Fig. 7 is the library quality inspection picture of bovine rumen tissue single-cell suspension obtained in the specific embodiment: according to the requirements of the sequencing platform, the size of the library is generally about 300-600bp, including sequencing joints, read1, read2, and 10X barcode , UMI, Poly DT, insert fragment and sample index; in a specific embodiment, it is judged qualified in the case of the main peak 450bp.

Fig. 8 is a flowchart of the method of the present invention.

detailed description

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

A bovine rumen epithelial tissue dissociation method for single-cell sequencing provided by the present invention is shown in Figure 8, and the specific steps are:

(1) Sample mechanical treatment: put a sterile RNase-free cell culture dish on an ice box, add 1 × PBS; pick bovine rumen tissue and place it on a culture dish, wash it twice with 1 × DPBS, and observe the residue in the muscle layer If the muscle layer is removed with scissors and tweezers, then the tissue block is cut into small pieces of 10×0.5 mm ² , transferred to an EP tube, added 20 mM EDTA twice the volume of the tissue, and soaked at 37°C for 30 min; then use 1×DPBS Wash 2 times, continue to cut the tissue to 1mm ³ , discard the supernatant, and pick an appropriate amount of tissue pieces into a new EP tube;

(2) Tissue enzymatic hydrolysis: Add 5ml 0.25% trypsin/EDTA to the EP tube containing the ^1mm3 tissue block in step (1), digest at 37°C for 5min; insert it into ice for 2min quickly after digestion, then add Volume pre-cooled HBSS, stop digestion; then centrifuge at 4°C, 300×g for 2min, then add 2ml HBSS, centrifuge at 300×g for 2min, wash again; then add 2ml of incubated enzyme dissociation solution (1.5mg/ ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I), incubate at 37°C for 30min; then directly add 2ml 10% FBS (V /V), inhibit enzyme activity;

(3) Filtration: Filter the cell suspension digested in step (2) with a stacked cell filter with a pore size of 30 and 70 μm, collect the filtered cell suspension in a new EP tube, centrifuge at 300×g, 4°C for 5 minutes , discard the supernatant.

(4) Cell washing: Add 2ml of HBSS to resuspend the cells in the EP tube containing the cell pellet in step (3) to wash the cells, centrifuge at 300×g, 4°C for 5 min, discard the supernatant; then add to the EP tube Resuspend the cells in 2ml 1×PBS (containing 0.04% BSA, that is, BSA with a concentration of 400mg/L) to wash the cells, centrifuge at 300×g, 4°C for 5min, discard the supernatant; then add 2ml 1×PBS ( containing 0.04% BSA) to resuspend the cells.

(5) Filtration again: filter the digested cell suspension in step (4) with a Miltenyi 20μm cell strainer, add 2ml 1×PBS (containing 0.04% BSA) to wash the filter membrane, and then filter at 300×g, 4°C Centrifuge for 5 minutes under the same conditions; observe the cell debris, if the debris rate is >10%, repeat this step;

(6) Cell observation and quality inspection: Gently blow the cell suspension obtained in step (5), take about 6 μL and place it in a hemocytometer, observe the background debris, cell clumping and cell density with a microscope; add trypan blue Staining, calculating cell viability, cell concentration and cell number;

(7) Dead cell removal (optional): If the cell activity observed in step (6) does not meet the cell activity requirements of single-cell sequencing, but the total amount of cells is sufficient, the dead cell removal device can be used to remove dead cells. First, place the dedicated MS or LS column, magnet, rack, and receiving tube on the ultra-clean bench to remove dead cells; in the ultra-clean bench, use double distilled water to dilute 20×Binding Buffer into 1×Binding Buffer (MS: 3-4ml/rxn or LS: 17-26ml/rxn); 300g, centrifuge at 4°C, discard the supernatant; collect the cell pellet, add 100μL Dead Cell Removal MicroBeads to mix well, incubate at room temperature for 15min; the incubation time is about 14min , add 1×Binding Buffer (MS: 500μl; LS: 3ml); after incubation, before the washing solution exposes the column, add 1×Binding Buffer (MS: 500-1000μl; LS: 1-10ml) to the cell suspension , and added to the top of the column; wash the column with 1×Binding Buffer (MS: 4×500μl; LS: 4×3ml); centrifuge the collection tube at 300×g, 4°C, discard the supernatant; then add an appropriate volume of Resuspend the cells in 0.04% BSA 1×PBS in 50-300 μmL (the amount added according to the amount of cell pellet); use a 1ml pipette gun to pipette and mix 5 times, continue to step (6) to observe until the total amount of cells and cell viability All meet the requirements.

Among the above dissociation steps of the bovine rumen tissue of the present invention, step (1) and step (2) are crucial to the high dissociation efficiency of the present invention.

The present invention also provides a high-efficiency method for the dissociation of bovine rumen tissue samples. The method includes that Ca ²⁺ and Mg ²⁺ are required to maintain the integrity of the tissue due to the tightness of the bovine rumen epithelial tissue, and EDTA can Absorb these ions from the living environment of these tissues, form chelates, promote the separation of cells, make the tissues soft, and make the enzyme dissociation solution added later act better on the cells.

The present invention also provides a tissue enzyme dissociation solution for single-cell dissociation of bovine rumen tissue samples. ml collagenase I (Worthington, A004214-0050), 1.5mg/ml collagenase IV (Worthington, A004210-0050), 100U/ml hyaluronidase (Worthington, A005477-0005), 50U/ml DNase I (Sigma, 11284932001).

The present invention also provides the application of the tissue enzyme dissociation solution in dissociation of bovine rumen tissue samples.

An example of the present invention is as follows:

Preparation before experiment

(1) Sterilize the sterile operating table for at least 30 minutes before the experiment;

(2) Weigh the specific enzyme or prepare the preservation solution of the enzyme;

(3) Prepare frozen plastic ice bags or ice boxes for use;

(4) Turn on the 37°C water bath, low-temperature centrifuge and ice maker in advance.

1. Mechanical separation of rumen tissue

(1) On a sterile operating table, place a sterile RNase-free cell culture dish on an ice box, and add 1×PBS;

(2) Immediately pick three healthy dairy cows with similar body conditions and production performance, collect tissues from the rumen abdominal sac, place them on a petri dish, wash them twice with 1×DPBS, and record them as sample 1, sample 2, and sample 3, as Each sample is subjected to subsequent experimental processing as parallel experiment (1), parallel experiment (2) and parallel experiment (3); Figure 1, Figure 2 and Figure 3 are the images of the single cell suspension of the three samples under the microscope : Less background debris, almost no cell agglomeration, moderate cell density;

(3) Observe the residual muscle layer, remove the muscle layer with sterilized surgical scissors and tweezers, then cut the tissue block into small pieces of ¹⁰ ×0.5mm2, transfer it to a 15ml EP tube, add 20mM EDTA submerged in the tissue, and Soak at 37°C for 30 minutes;

(4) Wash 2 times with 1×DPBS, continue to cut the tissue to 1mm ³ , discard the supernatant, and pick an appropriate amount of tissue pieces into a new 15ml EP tube;

2. Enzymatic hydrolysis of bovine rumen tissue

(1) Add 5ml of 0.25% trypsin/EDTA to the EP tube containing the ^1mm2 tissue block, and digest at 37°C for 5min;

(2) Immediately insert into ice for 2 minutes after digestion, then add an equal volume of pre-cooled HBSS to stop digestion; centrifuge at 4°C and 300g for 2 minutes, discard the supernatant;

(3) Add 2ml HBSS, centrifuge at 300×g for 2min, wash again, and discard the supernatant;

(4) Add 2ml of incubated enzyme dissociation solution (1.5mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I) , incubate at 37°C for 30min;

(5) Then directly add 2ml 10% FBS (V/V) to inhibit the enzyme activity;

3. Impurity filtration

Filter the digested cell suspension with stacked cell strainers with pore sizes of 30 and 70 μm, collect the filtered cell suspension in a new 15ml EP tube, centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant.

4. Cell washing

(1) Add 2ml HBSS to the EP tube containing the cell pellet, resuspend the cells to wash the cells, centrifuge at 300×g, 4°C for 5min, and discard the supernatant;

(2) Add 2ml of 1×PBS (containing 0.04% BSA) to the EP tube to resuspend the cells to wash the cells, centrifuge at 300×g, 4°C for 5min, and discard the supernatant;

(3) Then add 2ml 1×PBS (containing 0.04% BSA) to resuspend the cells.

5. Filter again

(1) Use Miltenyi 20μm cell filter to filter the digested cell suspension, add 2ml 1×PBS (containing 0.04% BSA) to wash the filter membrane, and then centrifuge at 300g, 4℃ for 5min;

(2) Observe the cell debris, if >10%, repeat this step;

6. Cell observation and quality inspection

(1) Gently blow out the cell suspension, take about 6 μL and place it in a hemocytometer, and observe the background fragments, cell clusters and cell density with a microscope; as shown in Figure 4 and Figure 5, they are parallel experiments (1) , (2) and (3) the cell concentration and the cell viability of bovine rumen tissue single cell suspension that obtain, sample one, two, three are respectively identical with the sample used in Fig. 1,2,3: cell concentration is respectively 1.15 ×10 ⁶ /mL, 1.27×10 ⁶ /mL and 2.64×10 ⁶ /mL, exceeding 10 ⁶ /mL; the cell viability was 88%, 94% and 94% respectively, reaching the standard of 85%;

(2) Add trypan blue staining, calculate cell viability, cell concentration and cell number;

7. Removal of dead cells (optional)

(1) First place the dedicated MS or LS column, magnet, shelf, and receiving tube on the ultra-clean bench to remove dead cells;

(2) In the ultra-clean bench, use double distilled water to dilute 20×Binding Buffer into 1×Binding Buffer (MS: 3-4ml/rxn or LS: 17-26ml/rxn);

(3) Then centrifuge at 300×g, 4°C, and discard the supernatant;

(4) Collect the cell pellet, add 100 μL Dead Cell Removal MicroBeads, pipette and mix well, incubate at room temperature for 15 minutes; add 1×Binding Buffer (MS: 500 μl; LS: 3ml) after the incubation time is about 14 minutes;

(5) After the incubation, before the rinse solution exposes the column, add 1×Binding Buffer (MS: 500-1000μl; LS: 1-10ml) to the cell suspension and add it to the top of the column;

(6) Use 1×Binding Buffer (MS: 4×500μl; LS: 4×3ml) to wash the column;

(7) Centrifuge the collection tube at 300×g, 4°C, and discard the supernatant;

(8) Then add an appropriate volume of 50-300 μmL (judging the amount added according to the amount of cell pellet) of 0.04% BSA1×PBS to resuspend the cells;

(9) Use a 1ml pipette gun to blow and mix 5 times, and continue to step (6) for observation until the total amount of cells and cell activity meet the requirements.

8. Quality inspection

In order to further verify the quality of the bovine rumen tissue single-cell suspension, the quality inspection of cDNA and library was carried out. As shown in Figure 6 and Figure 7, they are the cDNA quality inspection map and the library quality inspection map of the single-cell suspension of bovine rumen tissue respectively. The main peak of the cDNA quality inspection picture segment distribution map is greater than 1000bp and there is no jagged peak, which is judged as Passed; according to the requirements of the sequencing platform, the library size is generally around 300-600bp, including sequencing adapters, read1, read2, 10X barcode, UMI, Poly DT, insert fragments, and sample index; it is judged qualified when the main peak is 450bp.

The above-mentioned embodiments are used to illustrate the present invention, rather than to limit the present invention. Within the spirit of the present invention and the protection scope of the claims, any modification and change made to the present invention will fall into the protection scope of the present invention.

Claims

A bovine rumen epithelial tissue dissociation method for single-cell sequencing, characterized in that it specifically comprises the following steps:

(1) Mechanical separation of rumen tissue

Put a sterile RNase-free cell culture dish on an ice box and add PBS; put the rumen tissue in the culture dish, wash it with DPBS, use sterilized surgical scissors to remove the muscle layer, cut the tissue pieces into pieces, and put them in Soak in EDTA, then wash with DPBS again, continue to cut the tissue, and discard the supernatant;

(2) Enzyme digestion treatment

Add trypsin/EDTA to the last cut tissue piece in step (1) for digestion; after digestion, insert into ice and add HBSS to stop digestion; after centrifugation, wash again with HBSS; add the incubated enzyme dissociation solution, and the enzyme dissociation The solution contains neutral protease, collagenase I, collagenase IV, hyaluronidase and DNase I, after incubation, centrifuge; then add FBS to inhibit enzyme activity, and obtain a digested cell suspension;

(3) Impurity filtration

Filter the digested cell suspension in step (2) with a cell strainer and collect it into a new EP tube; discard the supernatant after centrifugation;

(4) Cell washing

Add HBSS to the cell pellet after centrifugation in step (3) to resuspend, discard the supernatant after centrifugation; add PBS containing BSA for resuspension, discard the supernatant after centrifugation; add PBS containing BSA to resuspend the cells to obtain a cell suspension liquid;

(5) filter again

Use a cell strainer to filter the cell suspension obtained in step (4), centrifuge after washing the filter membrane with PBS containing BSA; observe the cell debris, if the debris rate is greater than 10%, repeat this step;

(6) Cell observation and quality inspection

Blow evenly the cell suspension obtained in step (5), take part of the cell suspension and place it in a hemocytometer, observe the background debris, cell clumping and cell density with a microscope; add trypan blue staining, calculate cell viability, cell concentration and total cells. If it is observed that the cell activity does not meet the requirements for cell activity of single-cell sequencing, but the total number of cells is sufficient, the dead cell removal operation is required.
A method for dissociation of bovine rumen epithelial tissue for single-cell sequencing as claimed in claim 1, wherein if it is observed in step (6) that cell activity does not meet the requirements of single-cell sequencing for cell activity, but the cells When the total amount is sufficient, the dead cell removal device can be used to remove dead cells. The specific operation is as follows: In the ultra-clean bench, dilute 20×Binding Buffer with double distilled water to 1×Binding Buffer; centrifuge the cell suspension obtained in step (5) and discard the supernatant; collect the cell pellet, add Dead Cell Removal MicroBeads were blown and mixed; incubated at room temperature, during which the diluted Binding Buffer was added; after the incubation, before the washing solution exposed the column, the diluted Binding Buffer was added to the cell suspension and added to the top of the column; washed with the diluted Binding Buffer Column; centrifuge the collection tube and discard the supernatant; add BSA-containing PBS to resuspend the cells; mix by pipetting and continue to step (6) for observation.
A method for dissociation of bovine rumen epithelial tissue for single-cell sequencing as claimed in claim 1, wherein in step (1), firstly, the tissue block is cut into small pieces with a volume of 10×0.5mm 2 , when the tissue is continued to be chopped, the volume of the tissue block is 1mm 3 . The PBS is 1×PBS, and the amount added is 5 ml; the DPBS is 1×DPBS. The ratio of the volume of EDTA added to the volume of the tissue block is 2:1.
A bovine rumen epithelial tissue dissociation method for single-cell sequencing according to claim 1, characterized in that, in step (1), the EDTA concentration is 20 mM, and the method is soaked at 37° C. for 30 minutes.
A kind of bovine rumen epithelial tissue dissociation method for single-cell sequencing as claimed in claim 1, is characterized in that, in step (2), the concentration of described trypsin/EDTA is 2500mg/L, and the volume added is the same as The ratio of the tissue blocks after the tissue was further shredded was 2:1; the digestion conditions were as follows: the digestion temperature was 37° C., and the digestion time was 5 minutes. The cooling time in ice is 2 min. The volume of the HBSS is equal to that of the cell suspension, and the HBSS is pre-cooled HBSS; the centrifugation conditions are: the centrifugation speed is 300×g, the centrifugation temperature is 4° C., and the centrifugation time is 2 minutes. The ratio of the volume of the enzyme dissociation solution added to the tissue block after the tissue is continued to be shredded is 2:1, which contains 1.5mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I; the reaction conditions are: the incubation temperature is 37°C, and the incubation time is 30min. The FBS is 10% FBS (V/V), and the volume is the same as that of the enzyme dissociation solution added.
A bovine rumen epithelial tissue dissociation method for single-cell sequencing according to claim 1, characterized in that in step (3), the filtering uses stacked cell filters with pore sizes of 30 μm and 70 μm. The centrifugation conditions are as follows: the centrifugation speed is 300×g, the centrifugation temperature is 4° C., and the centrifugation time is 5 minutes.
A method for dissociation of bovine rumen epithelial tissue for single-cell sequencing according to claim 1, wherein in step (4), the amount of HBSS added is 2 mL. The amount of PBS added was 2 mL; the PBS was 1×PBS, and the PBS contained BSA at a concentration of 400 mg/L. The centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.
A kind of bovine rumen epithelial tissue dissociation method for single-cell sequencing as claimed in claim 1, is characterized in that, in described step (5), described cell filter uses Miltenyi 20 μ m cell filter; The PBS is 1×PBS, and the PBS contains BSA at a concentration of 400 mg/L. The centrifugation conditions are as follows: the centrifugation speed is 300 g, the centrifugation temperature is 4° C., and the centrifugation time is 5 min.
A method for dissociation of bovine rumen epithelial tissue for single-cell sequencing according to claim 2, wherein the device for removing dead cells includes MS or LS columns, magnets, shelves, and receiving tubes. For MS or LS, the amount of Binding Buffer used is different. The diluted Binding Buffer is 1×Binding Buffer; the usage condition of MS is 3-4ml/rxn, and the usage condition of LS is 17-26ml/rxn. The amount of Binding Buffer added during incubation is: add 500μl to MS, add 3ml to LS; after incubation, the amount of Binding Buffer added is: add 500-1000μl to MS; add 1-10ml to LS. When washing the column, the amount of Binding Buffer added is: 500μl each time in MS, 4 times in total; 3ml each time in LS, 4 times in total.
A bovine rumen epithelial tissue dissociation method for single-cell sequencing according to claim 2, wherein the amount of Dead Cell Removal MicroBeads added is 100 μl. The incubation time is 15 minutes, and the time of adding Binding Buffer is the 14th minute of incubation; the centrifugation conditions are: the centrifugation speed is 300×g, the centrifugation temperature is 4°C, and the centrifugation time is 5-10 minutes. The amount of PBS added is 50-300 μl; the PBS is 1×PBS, and the PBS contains BSA at a concentration of 400 mg/L.