GSM5324977: KO (knockout); Mus musculus; RNA-Seq
Single-cell suspensions were generated from adult WT and KO mouse testes using a two-step enzymatic digestion approach58. Mice were sacrificed, and then the testes were removed and decapsulated. The seminiferous tubules were cut into small pieces and incubated in DPBS containing 1 mg/ml Collagenase I (Gibco) at 37ºC for five minutes with gentle shaking. The dispersed seminiferous tubules and cells were collected by centrifugation at 100 g for two minutes at 4ºC. The pellet was washed once with DPBS and digested in 0.25% Trypsin (Gibco) containing 1 mg/ml DNase I (AppliChem) at 37ºC for five minutes with gentle shaking. The suspension was added with FBS (Vistech) to terminate digestion. Then, cells were collected by centrifugation at 600 g for five minutes and washed with 0.04% BSA/DPBS. The cells were then filtered through a 40-μm cell strainer (BD Falcon), and cell debris was removed by FACS. The cells were resuspended at a concentration of 1 million cells per ml in 0.04% BSA/DPBS. Chromium Next GEM Single-cell 3' GEM Library and Gel Bead Kit V3.1 (10× Genomics, 1000121) and Chromium Next GEM Chip G Single Cell Kit (10× Genomics, 1000120) were used for cell capture and cDNA synthesis. The cell suspension was loaded onto the Chromium single-cell controller (10× Genomics) to generate single-cell gelbead emulsions (GEMs) according to the manufacturer's instruction. About 20,000 cells were added to each channel, and the target recovery rate was estimated to be 13,000 cells. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed using a T series Multi-Block Thermal Cycler (LongGene). The following procedure was used: 53°C for 45 min, 85°C for 5 min, and 4°C forever. The quality of the generated cDNA was assessed by the Agilent 2100, which was performed by Emei Tongde (Beijing, China). Sequencing (performed by NovoGene, Beijing) was performed on the Illumina NovaSeq 6000 sequencer with 150 bp (PE150) paired-end reads.