GSM7438367
Wild-type and mutant testes were dissected and soaked in ice-cold PBS buffer. The tunica albuginea was carefully removed, and the seminiferous tubules were transferred to digestion buffer 1 (2mg/ml Collagenase I, 10% KSR in α-MEM). Subsequentially, the tissue was shaken to facilitate the dispersion and release the cell. The sample was then incubated in a 34°C water bath and rotate at 215 rpm for 30 min. Every 5 min, a wide pore pipette was used to gently pipette up and down five times. To release more single cells, the sample was then washed with 1X HBSS and immersed in digestion buffer 2 (700ug/ml trypsin dissolved in HBSS) for further digestion at 34°C and 215 rpm for another 10 minutes. The resulting cell suspension was filtered through a 40-µm cell strainer, washed with HBSS, and resuspended in 1ml of HBSS containing 0.04% BSA. The library was constructed with the 10x Single Cell 3' V3 kit and sequenced on one 2x150nt lane in a NovaSeq 6000 SP flowcell.