GSM4629902: AdId4eGFPbright-Rapa; Mus musculus; RNA-Seq
Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using  a FACS Aria (BD). Cells from adult Adult Id4-eGfp+ mouse were also enriched for spermatocytes and spermatids based on sedimentation velocity at unit gravity (Romrell et al., 1976; Bellve et al., 1977). Briefly, testis cells (10^6-10^7) suspended in 2 ml of buffer plus 0.5% BSA were loaded onto a 50 ml gradient of 2-4% BSA (McCarrey et al., 1992) and allowed to sediment for 2.5 hr at 4°C. Approximately one hundred 0.5 ml fractions were then collected in microcentrifuge tubes and analyzed for content of spermatocytes or spermatids on the basis of morphology under phase contrast optics (which typically yields =85% purity). Fractions containing spermatocytes or spermatids were pooled separately, concentrated (to ~2 x106 cells/ml) and stored in buffer containing FBS on ice until use. Ultimately, CD9bright/ID4-EGFP-birght testis cells from adult mice were used for single-cell RNA-seq facilitated by the 10x Genomics Chromium approach. Briefly, cell suspensions were loaded into Chromium microfluidic chips with 3' v3 chemistry and used to generate single-cell gelbead emulsions (GEMs) with using the Chromium controller (10x Genomics) per manufacturer recommendations (Zheng et al., 2017). In all cases, suspensions containing ~7000 cells were loaded on the instrument with the expectation of collecting up to 4,000 GEMs containing single cells. GEM-RT was performed in a T100 Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to manufacturer recommendations. Single-cell 3' libraries were prepared according to manufacturer recommendations (v3 chemistry).