GSM7051273: Yak_testis; Bos grunniens; RNA-Seq
The frozen testicular tissue was removed and immediately placed in a 37 ℃ water bath for 2 min. After the tissue was melted, take it out with tweezers, place it in the resuscitation solution (DMEM/F12 medium+0.5mol/L sucrose+20% FBS), place it in a 37 ℃ water bath for 2min, preheat the DPBS to wash the tissue block for 2-3 times, place the tissue block in DMEM/F12 medium (Gibco, Waltham, MA, USA), and cut the tissue with ophthalmic scissors. Transfer the testicular tissue of cutted to a 15mL centrifuge tube, and add a solution with 0.2% collagenase IV (2 mg/mL) (Sigma, City of Saint Louis, USA) and hyaluronidase (2 mg/mL) solution (Sigma, City of Saint Louis, USA) (1:1) of 8~10 times the total amount of tissue. The centrifuge tube was placed in a 37℃ constant temperature shaker at 120 rpm (30 min, after 15min, 200μL tissue digestive fluid was taken every 3min to observe the digestive condition under the microscope until the free tubules with no mass tissue could be clearly observed), centrifuge at 1000 r/min for 5 min and drain the supernatant. 0.25% trypsin solution (2.5 mg/mL) (Gibco, Waltham, MA, USA), 8-10 times the total amount of tissue blocks, was added again. Finally, the tissue digestible solution was placed in a constant temperature shaker at 37℃ at 120 rpm (40 min, after 15min, 200μL tissue digestible solution was taken every 3min to observe the digestion condition under the microscope until the single cell suspension could be clearly observed). Fetal bovine serum was added to terminate digestion. The mixed cell suspension was blown evenly and filtered through 70μm and 40μm screens successively. The filtrate was collected and centrifuged at 300×g 4℃ for 5 min, the supernatant was abandoned, and the cells were re-suspended in DMEM/F12 complete medium containing 10% FBS, and cultured at 37 ℃ with 5% CO2. Part of the cell suspension was stained with Trypan blue and counted by automatic counter to calculate the number of cells and viability. The concentration was ~1000 cells/uL and cell viability>90% were used for scRNA seq. The Cell suspension was loaded onto the Chromium Single Cell Controller instrument (10x Genomics, Pleasanton, CA, USA) and the bead and cells with Cell Barcode were wrapped in the droplet. The droplet containing cells was collected, and then the cells were lysed in the droplet, so that the mRNA in the cells combined with the bead Cell Barcode to form a Single Cell GEMs, and the reverse transcription reaction was performed in the droplet to construct the cDNA library. GEM-RT-PCR was performed with a 96-hole reaction module (Bio-Rad; CT022510) in the C1000 Touch thermal cycler to generate barcode cDNA using the following procedures: 53°C for 45 min; 85°C for 5 minutes; Maintain at 4°C. The sample index on the library sequence is used to distinguish the source of the target sequence.