GSM7466960: goat_male_E105, replicate1, scRNAseq; Capra hircus; RNA-Seq
For the collection of the testicular germ cells and somatic cells, testes were dissected under a dissection microscope and transferred to a 15 mL tube digested with 1 mg/mL collagenase IV, 1 mg/mL hyaluronidase, 0.25% trypsin, and 1 mg/mL DNase I for 30 min at 37C. To prepare the single-cell suspension for 10x Genomics single-cell RNA-seq, the cell suspension was repeatedly pipetted after adding serum, filtered through a 40 mm membrane, and resuspended in 0.1% serum/PBS. The cell suspension (1000 living cells per microliter determined by CountStar) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53C for 45 min, followed by 85C for 5 min, and hold at 4C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. According to the manufacture's introduction, Single-cell RNA-seq libraries were constructed using Single Cell 30 Library and Gel Bead Kit V3.1 The libraries were finally sequenced using an IlluminaNovaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).