CN113388570B - Bovine rumen epithelial tissue dissociation method for single cell sequencing - Google Patents
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Abstract
The invention discloses a bovine rumen epithelial tissue dissociation method for single cell sequencing, which comprises mechanical rumen tissue separation, enzyme digestion treatment, impurity filtration, cell cleaning, secondary filtration, cell observation and quality control, dead cell removal and the like. The method can obtain the rumen tissue single cell suspension with high activity, high cell number and low pollution, ensures the output of effective single cell number and the quality of single cell nucleic acid, and lays a foundation for single cell sequencing and data analysis; the invention solves the problem that the bovine rumen single cell dissociation technology is not systematically perfected in the prior art.
Description
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
Rumen is a specific digestive organ of ruminants such as dairy cows and the like, and plays an important role in digestion, metabolism, absorption and transportation of nutrient substances. Epithelial tissues are the main sites for rumen function, and how to maintain and improve the structural function of beef cattle and dairy cattle in breeding still faces serious challenges, such as subacute rumen acidosis and the like. Therefore, deeply analyzing the key physiological functions and the regulation mechanism of the rumen of the dairy cow becomes an important way for improving the health and the production performance of the dairy cow. The cell is the basic unit of life activity, the rumen epithelial tissue is positioned at the innermost side of the wall of the rumen, is a stratified squamous epithelium, and has a four-layer multifunctional cell layer structure, namely, a basal layer, a spinous layer, a granular layer and a cuticle are arranged in turn from the outermost side to the lumen of the rumen, but the research on the cell type and the molecular characteristics is obviously insufficient.
At present, rumen epithelial cells cultured at a tissue level or in vitro are mostly studied on the function of the rumen epithelial tissue of dairy cows. By analyzing transcriptome of rumen epithelial tissue, all expressed genes under specific conditions are captured, and then the change of system functions under different external treatments or stimulations is analyzed. However, 1g of ruminal epithelial tissue contains at least millions of cells, and traditional tissue level transcriptome analysis ignores cellular heterogeneity and fails to obtain functional features at the cellular level. In the cell level-based method, the purified cell suspension contains various types of cells such as basal layer, spinous layer and even granular layer cells, so that the existing research is built on a large class of epithelial cells and cannot be deeply researched into a single cell type, and the function of the rumen epithelial tissue of the dairy cow cannot be accurately analyzed at the cell level; meanwhile, a series of fundamental and important problems such as interaction between different types of cells of rumen epithelial tissues, functions of a small amount of mesenchymal cells, cell differentiation and development tracks and the like cannot be solved.
An important premise of single cell level research is to establish a tissue full dissociation and single cell suspension preparation method which accords with single cell capture tracking conditions; the greatest difference compared to rumen epithelial cell isolation culture methods is that such methods require as much dissociation of the tissue as possible to obtain all cell types and to capture and specifically track these cells with high efficiency. Previous researches also find that cell suspensions obtained by using the traditional rumen epithelial cell in-vitro separation and culture method cannot meet the requirement of single cell sequencing, and further exploration needs to be carried out on the method. The invention successfully establishes the rumen epithelial tissue dissociation and single cell suspension preparation technology for single cell sequencing such as a single cell transcriptome for the first time, and promotes the start of ruminant rumen single cell research. Meanwhile, a good foundation is established for developing other single cell-based researches such as single cell appearance groups, single cell proteome and the like in the future.
Disclosure of Invention
The invention aims to provide a bovine rumen epithelial tissue dissociation method for single cell sequencing, which aims to overcome the defects of the prior art, obtain a high-activity, high-cell-number and low-pollution rumen tissue single cell suspension, ensure the output of effective single cell number and the quality of single cell nucleic acid and facilitate single cell omics sequencing and data analysis.
The purpose of the invention is realized by the following technical scheme: a bovine rumen epithelial tissue dissociation method for single cell sequencing specifically comprises the following steps:
(1) Mechanical separation of rumen tissue
Placing a sterile cell culture dish without RNase on an ice box, and adding PBS; placing rumen tissue in a culture dish, cleaning DPBS, removing muscle layer with sterilized surgical scissors and forceps, cutting tissue blocks into pieces, soaking in EDTA, cleaning with DPBS again, cutting tissue into pieces, and removing supernatant;
(2) Enzyme digestion treatment
Adding pancreatin/EDTA into the tissue blocks cut at the end of the step (1) for digestion; after digestion, the mixture is inserted into ice and HBSS is added to stop digestion; centrifuging and then washing with HBSS; adding an incubated enzyme dissociation solution, wherein the enzyme dissociation solution comprises neutral protease, collagenase I, collagenase IV, hyaluronidase and DNase I, and centrifuging after incubation is finished; then adding FBS to inhibit enzyme activity to obtain digested cell suspension;
(3) Impurity filtering
Filtering the cell suspension digested in the step (2) by using a cell filter, and collecting the cell suspension to a new EP tube; centrifuging and then discarding the supernatant;
(4) Cell washing
Adding HBSS into the cell sediment after centrifugation in the step (3) for resuspension, and discarding the supernatant after centrifugation; adding PBS containing BSA for resuspension, centrifuging and then discarding the supernatant; adding PBS containing BSA to resuspend the cells again to obtain a cell suspension;
(5) Filtering again
Filtering the cell suspension obtained in the step (4) by using a cell filter, flushing the filter membrane by using PBS containing BSA, and centrifuging; observing the cell debris condition, and if the debris rate is more than 10%, repeating the step;
(6) Cell observation and quality control
Uniformly blowing the cell suspension obtained in the step (5), taking part of the cell suspension, placing the cell suspension in a blood counting chamber, and observing background fragments, cell clusters and cell density by using a microscope; trypan blue staining was added and cell viability, cell concentration and total cell amount were calculated. If the observed cell activity does not meet the requirement of single cell sequencing on cell activity, a dead cell removal operation is required when the total amount of cells is sufficient.
Further, if the cell activity observed in step (6) does not satisfy the requirement of single cell sequencing on cell activity, a dead cell removal device can be used to remove dead cells when the total amount of cells is sufficient. The specific operation is as follows: diluting 20 multiplied by Binding Buffer into 1 multiplied by Binding Buffer by double distilled water in a super clean bench; centrifuging the cell suspension obtained in the step (5) and then discarding the supernatant; collecting Cell precipitates, adding Dead Cell Removal MicroBeads, blowing, beating and uniformly mixing; incubating at room temperature, and adding diluted Binding Buffer during the incubation; after the incubation is finished and before the rinsing solution exposes the column, adding the diluted Binding Buffer into the cell suspension and adding the diluted Binding Buffer onto the column; washing the column with diluted Binding Buffer; centrifuging the collecting pipe and then discarding the supernatant; adding PBS containing BSA to resuspend the cells; and (5) blowing, beating and mixing uniformly, and continuing the observation in the step (6).
Further, in step (1), the tissue pieces are first cut into small pieces with a volume of 10X 0.5mm 2 When the tissue is continuously cut into pieces, the pieces are cut into pieces with the volume of 1mm 3 . The PBS is 1 XPBS, and the adding amount is 5ml; the DPBS is 1 XDPBS. The ratio of the volume of EDTA added to the volume of the tissue mass is 2:1.
further, in the step (1), the EDTA solution is soaked at a concentration of 20mM for 30min at a temperature of 37 ℃.
Further, in the step (2), the concentration of the pancreatin/EDTA is 2500mg/L, and the ratio of the added volume to the tissue mass after the tissue is continuously sheared into pieces is 2:1; the digestion conditions are as follows: the digestion temperature is 37 deg.C, and the digestion time is 5min. The cooling time in ice is 2min. The volume of the HBSS is equal to that of the cell suspension, and the HBSS is pre-cooled HBSS; the centrifugation conditions were: the centrifugation speed is 300 Xg, the centrifugation temperature is 4 ℃, and the centrifugation time is 2min. The ratio of the volume of the enzymolysis separation solution to the volume of the tissue blocks after the tissues are continuously cut into pieces is 2:1 comprising 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 as follows: the incubation temperature was 37 ℃ and the incubation time was 30min. The FBS was 10% FBS (V/V) in the same volume as the volume of added enzyme-eluted solution.
Further, in step (3), the filtration uses stacked cell filters of 30 μm and 70 μm pore size. The centrifugation conditions were: the centrifugation speed is 300 Xg, the centrifugation temperature is 4 ℃, and the centrifugation time is 5min.
Further, in step (4), the amount of HBSS added was 2mL. The addition amount of the PBS is 2mL; the PBS was 1 XPBS containing BSA at a concentration of 400 mg/L. The centrifugation conditions were all: the rotation speed of the centrifugation is 300g, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5min.
Further, in the step (5), the cell filter uses a beautiful and whirlwind 20 μm cell filter; the PBS was 1 XPBS containing BSA at a concentration of 400 mg/L. The centrifugation conditions were all: the rotation speed of the centrifugation is 300g, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5min.
Further, the cell killing device comprises an MS or LS column, a magnet, a frame and a receiving tube. For MS or LS, the using amount of the Binding Buffer is different, and the diluted Binding Buffer is 1 multiplied by the Binding Buffer; MS is used at 3-4ml/rxn, LS is used at 17-26ml/rxn. The adding amount of Binding Buffer during incubation is as follows: adding 500 mul into MS and 3ml into LS; after the incubation is finished, the adding amount of the Binding Buffer is as follows: adding 500-1000 μ l into MS; LS is added with 1-10ml. When the column is washed, the Binding Buffer is added in the following amount: 500 mul each time in MS, 4 times in total; 3ml in LS each time, 4 times.
Further, the addition amount of the Dead Cell Removal MicroBeads is 100. Mu.l. The incubation time is 15min, and the time for adding the Binding Buffer is 14min of incubation; the centrifugation conditions were all: the rotation speed of the centrifugation is 300 Xg, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5-10min. The addition amount of the PBS is 50-300 mu l; the PBS was 1 XPBS containing BSA at a concentration of 400 mg/L.
Compared with the prior art, the invention has the following beneficial effects: at present, a specific and systematic single cell dissociation method aiming at bovine rumen tissues does not exist, and the invention realizes a new breakthrough. The invention can obtain rumen tissue single cell suspension with high activity, high cell number, low pollution and complete cell types, and ensures the output of effective single cell number and the quality of single cell mRNA, thereby facilitating single cell sequencing and data analysis.
Drawings
FIG. 1 is an image under microscope of single cell suspension of bovine rumen tissue obtained from parallel experiment (1) in the embodiment: the background fragments are less, the cell agglomeration condition is almost zero, and the cell density is moderate;
FIG. 2 is an image under microscope of single cell suspension of bovine rumen tissue obtained from parallel experiment (2) in the embodiment: background fragments are less, cell agglomeration is almost avoided, and the cell density is moderate;
FIG. 3 is an image under microscope of single cell suspension of bovine rumen tissue obtained from parallel experiment (3) in the embodiment: background fragments are less, cell agglomeration is almost avoided, and the cell density is moderate;
FIG. 4 shows the cell concentrations of single cell suspensions of bovine rumen tissue obtained from experiments (1), (2) and (3) in parallel, wherein samples I, II and III are the same as those used in FIGS. 1, 2 and 3, respectively: the cell concentration was 1.15X 10 6 /mL，1.27×10 6 mL and 2.64X 10 6 /mL, more than 10 6 /mL；
FIG. 5 shows the cell viability of single cell suspensions of bovine rumen tissue obtained from experiments (1), (2) and (3) in parallel, in the embodiment, samples one, two and three are the same as the samples used in FIGS. 1, 2 and 3, respectively: the cell viability rates are respectively 88%,94% and 94%, and reach the standard of 85%;
FIG. 6 is a cDNA quality inspection diagram of single cell suspension of bovine rumen tissue obtained in the embodiment: judging the main peak value of the fragment distribution map to be qualified if the main peak value is larger than 1000bp and no jagged miscellaneous peak exists;
FIG. 7 is a library quality inspection chart of single cell suspension of bovine rumen tissue obtained in the embodiment: according to the requirement of a sequencing platform, the size of the library is generally about 300-600bp, wherein the size of the library comprises a sequencing joint, read1, read2, 10X barcode, UMI, poly DT, an insert and a sample index; in a specific embodiment, the pass is determined in the case of 450bp of the main peak.
FIG. 8 is a flow chart of the method of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention provides a bovine rumen epithelial tissue dissociation method for single cell sequencing, which comprises the following specific steps as shown in figure 8:
(1) Mechanical treatment of the sample: placing a sterile RNase-free cell culture dish on an ice box, and adding 1 XPBS; selecting rumen tissue of cattle, placing on culture dish, cleaning with 1 × DPBS for 2 times, observing residual condition of muscle layer, removing muscle layer with scissors and tweezers, and cutting tissue pieces into pieces of 10 × 0.5mm 2 Transferring the small pieces to an EP tube, adding 20mM EDTA with the volume 2 times that of the tissues, and soaking at 37 ℃ for 30min; then the tissue was further minced to 1mm using 2 washes of 1 XDPBS 3 Discarding the supernatant, and picking a proper amount of tissue blocks in a new EP tube;
(2) Tissue enzymolysis: to the step (1) contains 1mm 3 To an EP tube of the tissue mass was added 5ml of 0Digesting with 25% pancreatin/EDTA at 37 deg.C for 5min; quickly inserting into ice for 2min after digestion, then adding HBSS precooled in the same volume, and stopping digestion; centrifuging at 4 deg.C and 300 × g for 2min, adding 2ml HBSS, centrifuging at 300 × g for 2min, and cleaning; then adding incubated 2ml of enzymolysis dissociation solution (1.5 mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase and 50U/ml DNase I), and incubating for 30min at 37 ℃; then, 2ml of 10% FBS (V/V) was directly added to inhibit the enzyme activity;
(3) And (3) filtering: filtering the digested cell suspension of step (2) with a 30 and 70 μm pore size stacked cell filter, collecting the filtered cell suspension in a new EP tube, centrifuging at 300 Xg and 4 ℃ for 5min, and discarding the supernatant.
(4) Cell washing: adding 2ml of HBSS to the EP tube containing the cell sediment in the step (3) to resuspend the cells so as to clean the cells, centrifuging the mixture at 300 Xg and 4 ℃ for 5min, and removing the supernatant; then 2ml of 1 XPBS (containing 0.04% BSA, i.e., BSA at a concentration of 400 mg/L) was added to the EP tube to resuspend the cells and wash the cells, and the cells were centrifuged at 300 Xg at 4 ℃ for 5min, and the supernatant was discarded; then, 2ml of 1 XPBS (containing 0.04% BSA) was added to resuspend the cells.
(5) And (3) filtering again: filtering the cell suspension digested in step (4) with a Meitian and whirly 20 μm cell filter, washing the filter with 2ml of 1 XPBS (containing 0.04% BSA), and centrifuging at 300 Xg, 4 ℃ for 5min; observing the cell debris condition, and if the debris rate is more than 10%, repeating the step;
(6) Cell observation and quality inspection: uniformly blowing the cell suspension obtained in the step (5) gently, placing about 6 mu L of the cell suspension in a blood counting chamber, and observing background fragments, cell clusters and cell density by using a microscope; adding trypan blue for staining, and calculating the cell viability, the cell concentration and the cell number;
(7) Dead cell removal (selection): if the cell activity observed in step (6) does not meet the requirement of single cell sequencing on cell activity, a dead cell removing device can be used to remove dead cells when the total amount of cells is sufficient. Firstly, placing a special MS or LS column, a magnet, a frame and a receiving pipe on a super clean bench to prepare for removing dead cells; diluting 20 XBinding Buffer to 1 XBinding Buffer (MS: 3-4ml/rxn or LS:17-26 ml/rxn) in a super clean bench by using double distilled water; 300g, centrifuging at 4 deg.C, and removing supernatant; collecting Cell precipitate, adding 100 μ L of Dead Cell Removal MicroBeads, blowing, mixing, and incubating at room temperature for 15min; after an incubation time of about 14min, 1 × Binding Buffer (MS: 500. Mu.l; LS:3 ml) was added; after the incubation is finished, 1 XBinding Buffer (MS: 500-1000. Mu.l; LS:1-10 ml) is added to the cell suspension before the rinse exposes the column and is added on the column; the column was washed with 1 XBinding Buffer (MS: 4X 500. Mu.l; LS: 4X 3 ml); centrifuging the collection tube at 300 Xg and 4 deg.C, and discarding the supernatant; then adding an appropriate volume of 50-300. Mu.mL (the amount added is judged according to the amount of cell pellet) of 0.04% BSA1 × PBS to resuspend the cells; and (5) uniformly mixing the cells by blowing and beating the cells by using a 1ml pipette gun for 5 times, and continuing the observation in the step (6) until the total amount and the activity of the cells meet the requirements.
In the bovine rumen tissue dissociation steps, the step (1) and the step (2) are crucial to the invention for obtaining high-efficiency dissociation efficiency.
The present invention also provides an efficient method for bovine rumen tissue sample dissociation, which comprises that Ca is required for survival due to the compactness of bovine rumen epithelial tissue 2+ And Mg 2+ The integrity of the tissues can be maintained, EDTA can absorb the ions from the living environment of the tissues to form a chelate, so that the mutual separation of cells is promoted, the tissues are soft, and the subsequently added enzymolysis chaotropic liquid can better act on the cells.
The invention also provides a cathepsin dissociation solution for bovine rumen tissue sample single cell dissociation, which comprises 1.5mg/ml neutral protease (Worthington, A02100-0010), 1.5mg/ml collagenase I (Worthington, A004214-0050), 1.5mg/ml collagenase IV (Worthington, A004210-0050), 100U/ml hyaluronidase (Worthington, A005477-0005) and 50U/ml DNase I (Sigma, 11284932001).
The invention also provides application of the cathepsin dissociation liquid in dissociation of bovine rumen tissue samples.
One embodiment of the invention is as follows:
preparation before experiment
(1) Sterile worktop was sterilized for at least 30 minutes prior to the experiment;
(2) Weighing specific enzyme or enzyme-prepared preservation solution;
(3) Preparing a frozen plastic ice bag or ice box for later use;
(4) The 37 ℃ water bath, the low-temperature centrifuge and the ice maker were opened in advance.
1. Mechanical separation of rumen tissue
(1) Placing a sterile cell culture dish without RNase on an ice box on a sterile operating platform, and adding 1 XPBS;
(2) Then, selecting three healthy cows with similar body conditions and production performance, collecting tissues in a rumen abdominal sac, placing the tissues on a culture dish, cleaning the tissues 2 times by using 1 XDPBS, marking the tissues as a first sample, a second sample and a third sample, and carrying out subsequent experimental treatment on each sample to be used as a parallel experiment (1), a parallel experiment (2) and a parallel experiment (3); fig. 1, 2 and 3 are images under a microscope of single cell suspensions of three samples, respectively: background fragments are less, cell agglomeration is almost avoided, and the cell density is moderate;
(3) Observing residual condition of muscular layer, removing muscular layer with sterilized surgical scissors and forceps, and cutting tissue pieces into pieces of 10 × 0.5mm 2 Transferring the small blocks into a 15ml EP tube, adding 20mM EDTA which has been immersed in the tissue, and soaking at 37 ℃ for 30min;
(4) The tissue was further minced to 1mm using 2 washes of 1 XDPBS 3 Discarding the supernatant, and picking a proper amount of tissue blocks in a new 15ml EP tube;
2. enzymolysis of bovine rumen tissue
(1) To contain 1mm 2 Adding 5ml of 0.25% pancreatin/EDTA into an EP tube of the tissue block, and digesting for 5min at 37 ℃;
(2) Quickly inserting into ice for 2min after digestion, then adding HBSS precooled in the same volume, and stopping digestion; centrifuging at 4 deg.C and 300g for 2min, and removing supernatant;
(3) Adding 2ml HBSS, centrifuging at 300 Xg for 2min, cleaning again, and discarding the supernatant;
(4) Adding incubated 2ml enzyme dissociation solution (1.5 mg/ml neutral protease, 1.5mg/ml collagenase I, 1.5mg/ml collagenase IV, 100U/ml hyaluronidase, 50U/ml DNase I), and incubating at 37 deg.C for 30min;
(5) Then, 2ml of 10% FBS (V/V) was directly added to inhibit the enzyme activity;
3. impurity filtering
The digested cell suspension was filtered through a 30 and 70 μm pore size stack of cell filters, the filtered cell suspension was collected in a new 15ml EP tube, centrifuged at 300 Xg for 5min at 4 ℃ and the supernatant discarded.
4. Cell washing
(1) Adding 2ml HBSS into EP tube containing cell precipitate, resuspending cells to clean cells, centrifuging at 300 Xg and 4 deg.C for 5min, and discarding supernatant;
(2) Then 2ml of 1 XPBS (containing 0.04% BSA) was added to the EP tube to wash the resuspended cells, and the cells were centrifuged at 300 Xg and 4 ℃ for 5min, and the supernatant was discarded;
(3) Then, 2ml of 1 XPBS (containing 0.04% BSA) was added to resuspend the cells.
5. Filtering again
(1) Filtering the digested cell suspension with a Meitian whirlpool 20 μm cell filter, washing the filter with 2ml of 1 XPBS (containing 0.04% BSA), and centrifuging at 300g at 4 ℃ for 5min;
(2) Observing cell debris, and if the cell debris is greater than 10%, repeating the step;
6. cell observation and quality inspection
(1) Gently blowing the cell suspension evenly, placing about 6 mu L of the cell suspension in a blood counting chamber, and observing background debris, cell clusters and cell density by using a microscope; as shown in fig. 4 and fig. 5, the cell concentration and cell viability rate of the single cell suspension of bovine rumen tissue obtained in parallel experiments (1), (2) and (3) were the same as those in fig. 1, 2 and 3: the cell concentration was 1.15X 10 6 /mL，1.27×10 6 mL and 2.64X 10 6 /mL, more than 10 6 Per mL; the cell viability rates are respectively 88%,94% and 94%, and reach the standard of 85%;
(2) Trypan blue is added for staining, and the cell viability, the cell concentration and the cell number are calculated;
7. dead cell removal (optional)
(1) Firstly, placing a special MS or LS column, a magnet, a frame and a receiving pipe on a super clean bench to prepare for removing dead cells;
(2) Diluting 20 × Binding Buffer to 1 × Binding Buffer (MS: 3-4ml/rxn or LS:17-26 ml/rxn) in a super clean bench using double distilled water;
(3) Centrifuging at 300 Xg and 4 deg.C, and removing supernatant;
(4) Collecting Cell precipitate, adding 100 μ L of Dead Cell Removal MicroBeads, blowing, mixing, and incubating at room temperature for 15min; after an incubation time of about 14min, 1 XBinding Buffer (MS: 500. Mu.l; LS:3 ml) was added;
(5) After the incubation is finished and before the rinsing solution exposes the column, 1 × Binding Buffer (MS: 500-1000 μ l; LS:1-10 ml) is added to the cell suspension and added on the column;
(6) The column was washed with 1 XBinding Buffer (MS: 4X 500. Mu.l; LS: 4X 3 ml);
(7) Centrifuging the collection tube at 300 Xg and 4 deg.C, and discarding the supernatant;
(8) Then adding a suitable volume of 50-300 μmL (the amount added is judged according to the amount of cell pellet) of 0.04% BSA1 × PBS to resuspend the cells;
(9) And (5) uniformly mixing the cells by blowing and beating the cells by using a 1ml pipette gun for 5 times, and continuing the observation in the step (6) until the total amount and the activity of the cells meet the requirements.
8. Quality inspection
In order to further verify the quality of the obtained bovine rumen tissue single cell suspension, cDNA and library quality tests are carried out. As shown in fig. 6 and 7, the cDNA quality inspection map and library quality inspection map of bovine rumen tissue single cell suspension are respectively, the main peak value of the fragment distribution map of the cDNA quality inspection map is greater than 1000bp and no jagged miscellaneous peak, and the determination is passed; according to the requirements of a sequencing platform, the size of the library is generally about 300-600bp, wherein the size of the library comprises a sequencing joint, read1, read2, 10X barcode, UMI, poly DT, an insert and a sample index; the peak was judged to be 450 bp.
The above-described embodiments are intended to illustrate rather than limit the invention, and any modifications and variations of the present invention are within the spirit and scope of the appended claims.
Claims (4)
1. A bovine rumen epithelial tissue dissociation method for single cell sequencing is characterized by comprising the following steps:
(1) Mechanical separation of rumen tissue
Placing a sterile cell culture dish without RNase on an ice box, and adding PBS; placing rumen tissue in culture dish, cleaning DPBS, removing muscle layer with sterilized surgical scissors forceps, and cutting tissue pieces into pieces with volume of 10 × 0.5mm 2 Soaking in EDTA, cleaning with DPBS, and cutting tissue until the volume of tissue block is 1mm 3 Discarding the supernatant; the PBS is 1 XPBS, and the adding amount is 5ml; the DPBS is 1 XDPBS; the ratio of the volume of EDTA added to the volume of the tissue mass is 2:1; the concentration of the EDTA is 20mM, and the EDTA is soaked for 30min at the temperature of 37 ℃;
(2) Enzyme digestion treatment
Adding pancreatin/EDTA into the tissue blocks cut at the end of the step (1) for digestion; the concentration of the pancreatin/EDTA is 2500mg/L, the ratio of the added volume to the tissue mass after the tissue is continuously cut is 2:1; the digestion conditions are as follows: the digestion temperature is 37 ℃, and the digestion time is 5min; after digestion, the mixture is inserted into ice and HBSS is added to stop digestion; cooling in ice for 2min; the volume of the HBSS is equal to that of the cell suspension, and the HBSS is precooled HBSS; washing with HBSS again after centrifugation; adding an incubated enzyme dissociation solution, wherein the ratio of the volume of the added enzyme dissociation solution to the volume of the tissue mass after the tissue is continuously sheared into pieces is 2:1 comprising 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 as follows: incubation temperature is 37 ℃, and incubation time is 30min; the FBS was 10% FBS (V/V) in the same volume as the volume of the added enzyme-dissociating solution; centrifuging after the incubation is finished; then adding FBS to inhibit enzyme activity to obtain digested cell suspension; the centrifugation conditions were: the centrifugal speed is 300 Xg, the centrifugal temperature is 4 ℃, and the centrifugal time is 2min;
(3) Impurity filtering
Filtering the cell suspension digested in the step (2) by using a cell filter, and collecting the cell suspension to a new EP tube; centrifuging and then discarding the supernatant; the filtration uses stacked cell filters of 30 μm and 70 μm pore size; the centrifugation conditions were: the centrifugal speed is 300 Xg, the centrifugal temperature is 4 ℃, and the centrifugal time is 5min;
(4) Cell washing
Adding HBSS into the cell sediment after centrifugation in the step (3) for resuspension, and discarding the supernatant after centrifugation; the adding amount of the HBSS is 2mL; adding PBS containing BSA for resuspension, centrifuging and then discarding the supernatant; adding PBS containing BSA to resuspend the cells again to obtain a cell suspension; the adding amount of the PBS is 2mL; the PBS is 1 XPBS, and the PBS contains BSA with the concentration of 400 mg/L; the centrifugation conditions were all: the rotating speed of the centrifugation is 300g, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5min;
(5) Filtering again
Filtering the cell suspension obtained in the step (4) by using a cell filter, flushing the filter membrane by using PBS containing BSA, and centrifuging; observing the cell debris condition, and if the debris rate is more than 10%, repeating the step; the cell filter uses a beautiful and whirlwind 20 mu m cell filter; the PBS is 1 XPBS, and the PBS contains BSA with the concentration of 400 mg/L; the centrifugation conditions were as follows: the rotating speed of the centrifugation is 300g, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5min;
(6) Cell observation and quality inspection
Uniformly blowing the cell suspension obtained in the step (5), taking part of the cell suspension, placing the cell suspension in a blood counting chamber, and observing background fragments, cell clusters and cell density by using a microscope; adding trypan blue for staining, and calculating cell activity, cell concentration and cell total amount; if the observed cell activity does not meet the requirement of single cell sequencing on cell activity, a dead cell removal operation is required when the total amount of cells is sufficient.
2. The bovine rumen epithelial tissue dissociation method for single cell sequencing according to claim 1, wherein if the cell activity observed in step (6) does not satisfy the cell activity requirement of single cell sequencing, but the total amount of cells is sufficient, a dead cell removal device can be used for dead cell removal; the specific operation is as follows: diluting 20 multiplied Binding Buffer into 1 multiplied Binding Buffer by double distilled water in a super clean bench; centrifuging the cell suspension obtained in the step (5) and then discarding the supernatant; collecting Cell precipitates, adding Dead Cell Removal MicroBeads, blowing, beating and uniformly mixing; incubating at room temperature, and adding diluted Binding Buffer during the incubation; after the incubation is finished and before the rinsing solution exposes the column, adding the diluted Binding Buffer into the cell suspension and adding the diluted Binding Buffer onto the column; washing the column with diluted Binding Buffer; centrifuging the collecting pipe and then discarding the supernatant; adding PBS containing BSA to resuspend the cells; and (5) blowing, beating and mixing uniformly, and continuing the observation in the step (6).
3. The bovine rumen epithelial tissue dissociation method for single cell sequencing according to claim 2, wherein the de-dead cell device comprises MS or LS columns, magnets, shelves, receiving tubes; for MS or LS, the use amount of the Binding Buffer is different, and the diluted Binding Buffer is 1 multiplied by the Binding Buffer; the using condition of MS is 3-4ml/rxn, and the using condition of LS is 17-26ml/rxn; the adding amount of Binding Buffer during incubation is as follows: adding 500 mul into MS and 3ml into LS; after the incubation is finished, the adding amount of the Binding Buffer is as follows: adding 500-1000 μ l into MS; adding 1-10ml of LS; when the column is washed, the Binding Buffer is added in the following amount: 500 mul each time in MS, 4 times in total; 3ml in LS each time, 4 times.
4. The method for bovine rumen epithelial tissue dissociation for single Cell sequencing of claim 2, wherein said Dead Cell Removal MicroBeads are added in an amount of 100 μ l; the incubation time is 15min, and the time for adding the Binding Buffer is 14min of incubation; the centrifugation conditions were all: the rotation speed of the centrifugation is 300 Xg, the temperature of the centrifugation is 4 ℃, and the time of the centrifugation is 5-10min; the addition amount of the PBS is 50-300 mu l; the PBS was 1 XPBS containing BSA at a concentration of 400 mg/L.
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