10x Genomics single-cell RNA-seq

Through the UTSA Genomics Core, suspensions of ~6,400 ID4-EGFP^bright mouse spermatogonia were loaded into Chromium micro-fluidic chips with 3′ v3 chemistry and used to generate single-cell gelbead emulsions (GEMs) for ~4,000 single cells using the Chromium controller (10x Genomics) per manufacturer recommendations (Zheng et al., 2017). Reverse transcription was performed in a T100 Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to manufacturer recommendations (User Guide {"type":"entrez-nucleotide","attrs":{"text":"CG000204","term_id":"33868852","term_text":"CG000204"}}CG000204 Rev D). Libraries were sequenced on an Illumina NovaSeq instrument (NovoGene, Inc.) using PE150 chemistry. Trimmed FASTQ files [Read 1 - (28bp Cell barcode & UMI), 10bp i7 index, and Read2 - 90bp) were generated using CellRanger mkfastq. Primary data analysis (alignment, filtering, and UMI counting) to determine transcript counts per cell (producing a gene-barcode matrix), quality control, clustering and statistical analysis were performed using CellRanger v3.1.0 (10x Genomics) using default settings and a customized GRCm38 (mouse mm10) reference dataset containing the EGFP cDNA sequence (see Key resources table). Quality control metrics from these new single-cell data are found in Table S6.