GSM7233605: wild-type P15 testes, biological replicate 2, scRNAseq; Mus musculus; RNA-Seq
Single-cell sequencing libraries were prepared and sequenced in two batches, with each batch containing one P15 littermate pair comprised of one wild-type and one Meioc-null pup.  One mouse testis per P15 pup was enzymatically dissociated by first shaking vigorously for 30 seconds and incubating at 35˚C for 7 minutes in 900 ul of 0.1% collagenase type I (Worthington LS004196) in Hank's Balanced Salt Solution (HBSS), supplemented with TURBO DNase (ThermoFisher Scientific AM2238) for a final concentration of 2U/mL. After allowing the sample to sit for 1 minute such that the seminiferous tubules settled to the bottom of the tube, the interstitium was removed by discarding the resulting supernatant, and the remaining sample was incubated at 35˚C for 25 minutes (with gently pipetting every 5 minutes) in 800 ul of 0.05% trypsin and 0.1% collagenase type I in HBSS, supplemented with TURBO DNase for a final concentration of 2U/mL. The cell suspension was then passed through a 40um nylon strainer, and cell suspension was allowed to digest for 35˚C for an additional 20 minutes. The digestion was quenched by adding 200ul fetal bovine serum (FBS) and gently mixing. The cell suspension was again passed through a 40um nylon strainer. The concentration of the resulting cell suspension was assessed using trypan blue on the Countess Cell Counter. Cells were pelleted at 500g for 4 minutes at room temperature, resuspended in 0.05% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for a target concentration of 1000 cells per microliter, and placed on ice. Viability and concentration were re-assessed ~20 minutes prior to loading on the Chromium Controller using trypan blue on a Countess Cell Counter, at which point all samples had a minimum of 90% viability. Cell suspensions were loaded onto the Chromium Controller, aiming for recovery of 10,000 cells per sample. Libraries were generated using the Chromium Next GEM Single Cell 3ʹ v3.1 (10x Genomics), according to manufacturer's instructions