title: GSM5901244: ID_Neonates2; Mus musculus; RNA-Seq
library_construction_protocol: Testicular cells were collected from 6-8 day (n=3) and 4-month-old mouse testes (n=2)  by 2 step enzymatic digestion as previously described (Yeh et al., 2007). Spermatogonia from 6-8 old mice were then isolated by size exclusion and granularity using FACS light scatter properties. The primary contaminating cell type in the enriched spermatogonia population were somatic peritubular myoid and Sertoli cells. Spermatogonia from 4-month-old  were isolated by fluorescence assisted cell sorting (FACS) (FACSAria II, BD Biosciences), using antibodies against GFR⍺1 ([1:100] Neuromics, goat polyclonal) and cKIT ([1:100] AbCam, rat polyclonal). The primary contaminating cell type in the enriched spermatogonia population after FACS was further differentiating germ cells (spermatocytes and round spermatids).   Isolated primary murine spermatogonia samples were resuspended in PBS supplemented with 1% BSA and 6000–10,000 cells per sample were processed using ChromiumTM Single Cell 3′ v2 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Samples were sequenced on an Illumina HiSeq4000 with paired-end 50 base pair reads by Genome Quebec (Montreal, QC, Canada). Isolated primary murine spermatogonia samples were resuspended in PBS supplemented with 1% BSA and 6000–10,000 cells per sample were processed using ChromiumTM Single Cell 3′ v2 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Samples were sequenced on an Illumina HiSeq4000 with paired-end 50 base pair reads by Genome Quebec (Montreal, QC, Canada). Briefly, single cells were partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction.