Type 1

Purified undifferentiated spermatogonia for RNA-seq were obtained following the 3S protocol (Romer et al., 2018 (link)). Germ line lineage sorting was carried out with the Ddx4^Cre and ROSA26^tdTomato alleles, which contains a loxP-STOP-loxP-tdTomato construct. Via this genetic strategy, Cre recombinase was specifically expressed in germ cells, where it excised the STOP codon and activated tdTomato protein expression. After synchronization of spermatogenesis with WIN 18,446, testes were collected on P9 and biopsied for histological analysis. For the remaining testis pair, the tunica albuginea was removed, and the tissue was dissociated into a single-cell suspension using collagenase, type I (Worthington Biochemical LS004196) and trypsin as previously described (Romer et al., 2018 (link)) with one modification: TURBO DNAse (Thermo Fisher Scientific AM2238) was used in place of DNAse I. DAPI was added to cells prior to cell sorting on a FACSAria II (BD Biosciences). Single cells were gated based on forward and side light scatter, and high DAPI-positive (dead) cells were excluded. The undifferentiated spermatogonia were isolated based on tdTomato fluorescence and sorted in 1% BSA in PBS. Cells were transferred into and stored at −80 °C in Trizol LS (Thermo Fisher Scientific).