MAX controls meiotic entry in sexually undifferentiated germ cells
In mammals, female, but not male, germ cells onset meiosis physiologically during embryonic stages. We examined whether germ cell-specific disruption of Max gene leads to the activation of meiosis-related genes in embryonic male germ cells by single cell RNA sequencing. Overall design: Single-cell RNA sequencing was carried out on gonadal cells from 6 Max cKO embryos at E12.5. These embryos included 4 with the Max fl/fl genotype driven by the Oct4-CreERT2 transgene and 2 controls with the Max fl/fl genotype. The pregnant mice from which these embryos were derived had been administered tamoxifen at E8.5. Each set of gonads obtained at E12.5 was individually dissociated using typsin-EDTA into single-cell suspensions. Cells from gonads with the CreERT2 transgene were then combined with those lacking the gene for the sequencing analysis. Library preparation, including the creation of gel beads and barcoding, was done using the Single Cell 3' Reagent Kits v3 (10X Genomics). Sequencing was performed on an Illumina HiSeq platform, aiming for >50,000 reads per cell across 6,000 cells. The reference genome employed was the Genome Reference Consortium Mouse Build 38 (mm10), supplemented with a general transfer format file (Ensembl 93). Additional sequences for the CreERT2 cDNA and the SV40 genome were incorporated into the reference genome, labeled as ENSG00000N1 and ENSG00000N2, respectively. Data analysis was executed using Cell Ranger (v7.0.1) and the Loupe Browser (6.5.0).