GSM8676538: Wild-type testes, rep1, scRNA-seq; Mus musculus; RNA-Seq
To prepare germ cells for FACS sorting, a single cell suspension from testes was obtained following the method described by Ascenção et al.Testes from 2-5 mice were collected, the tunica albuginea was removed, and tubules were placed in 10 ml of preheated (37°C) DMEM-F12 containing 2 mg of Collagenase 1A (Sigma) for 2 minutes with manual shaking. Digestion was stopped by two washes of the tubules with DMEM-F12. Next, the tubules were incubated in 10 ml of DMEM-F12 with 5 mg of trypsin (Sigma) and 7 mg/ml of DNAse I (Sigma) at 37°C with constant shaking (150 rpm) until the length of the tubules was about 1 mm (approximately 10 minutes). Digestion was stopped by addition of 3 mls of Fetal Bovine Serum (FBS, Sigma) and 30 ml of DMEM F-12. Digested tubules were strained on a 100 μm strainer and centrifuged for at 15°C for 5 minutes at 600g. The single cell suspension of germ cells obtained was then resuspended in FBS and preserved at 37°C until fluorescent activated cell sorting (FACS). Germ cell single cell suspension was incubated with 10 μM of Vybrant dye cycle (VDG) (Invitrogen) for 30 min at 37°C in the dark and constant rocking.  Sorting of cells was performed at Cornell Flow Cytometer Facility, using a Sony MA900 fluorescent activated cell sorter, tuned to emit at 488 nm and laser power set to 100 mW to collect for VDG positive cells. Flow cytometric profiles were obtained by representing Forward Scatter (Y axes) vs VDG fluorescence intensity (X axes), to identify different cell populations in the sample based on their DNA content and size. To enrich for spermatocytes for single cell RNA-seq, a sorting gate was set to collect all germ cell types excluding sperm. Single cell RNA sequencing libraries were prepared at the Transcriptional Regulation and Expression Facility at Cornell using the 10X Genomics Chromium Single Cell 3′ RNA-seq v3.1 kit. Flow sorted cells were processed on the 10X Genomics Chromium System, targeting a total of 7000 cells per sample. Quality control was evaluated using an ABI DNA Fragment Analyzer