Single-Cell Testis Dissociation for scRNA-seq

The testis samples from each individual were cut into smaller pieces with the volume less than 0.5 mm³ after washing three times with PBS, then subjected to enzymatic digestion comprising Collagenase IV (1 mg/mL) and Dnase I (1 μg/μL) in a 1.5 mL centrifuge tube at 37 °C for 15 min as described previously [16 (link), 26 (link)]. During the digestion period, the tissues were mixed by pipetting up and down once in every five minutes. Enzymic digestion was terminated by adding 10% FBS (in DMEM) and testicular single cells were obtained by filtering through a 40-μm nylon mesh. After centrifugation at 1500 rpm for 5 min at 4 °C, the supernatant material was removed and the single cells were washed with 0.04% BSA DPBS for three times and the cell number and cell viability were measured by using the Countess® II Automated Cell Counter. The cells were resuscitated to a concentration of 700–1200 cells/μL with a viability ≥ 85%, ready for scRNA-seq.