title: GSM3302524: AdultHuman-Spermatocytes_17-11; Homo sapiens; RNA-Seq
library_construction_protocol: Suspensions of human seminiferous tubule cells were                     prepared from adult testicular tissue parenchyma by a two-step enzymatic                     digestion was as described previously (Hermann et al., 2007b; Hermann et al.,                     2009; Dovey et al., 2013). Briefly, testis tissue was digested with 2mg/ml                     collagenase type IV at 37oC with vigorous agitation, washed with HBSS to remove                     interstitial cells, followed by digestion with 0.25% trypsin/EDTA containing                     1.4mg/ml DNase I for 15 minutes at 37oC with trituration every 5 minutes, and                     quenched with 10% FBS. In all cases, cell suspensions were strained (40um pore                     size for mouse, 70um pore size for humans) and suspended in MEMa containing 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 106 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS)                     containing 10% FBS (DPBS+S), labeled with antibodies (see Key Resources Table),                     and subjected to FACS using a FACS Aria (BD). Positive antibody labeling was                     determined by comparison to staining with isotype control antibodies. Human                     spermatogonia were isolated by sorting cells with the phenotype HLA-ABCnegative                     / CD49enegative / THY1dim / ITGA6+ / EpCAMdim dim. These markers were chosen                     because previous results indicate they specifically label undifferentiated                     spermatogonia, including some with colonization potential based on                     xenotransplantation (Dovey et al., 2013; Altman et al., 2014; Smith et al.,                     2014; Valli et al., 2014). Adult Human testis cells 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, sorted spermatoginia,Sta-Put-enriched                     spermatocytes and spermatids, or unselected spermatogenic cells from adult human                     testicular tissue were used for single-cell RNA-seq facilitated by the 10X                     Genomics Chromium Drop-Seq approach. Briefly, Cell suspensions were loaded into                     Chromium microfluidic chips with 3' v2 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 ~8700 cells were loaded on the instrument with the                     expectation of collecting up to 5,000 GEMs containing single cells. For the                     multiplet test, equal numbers of mouse and human cells were loaded. 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 (v2 chemistry).