Transcription factor NFYA directs male meiotic entry by regulating accessible chromatin at meiotic promoters in mice
Transcription factor NFYA directs male meiotic entry by regulating accessible chromatin at meiotic promoters in mice
FACS-purified spermatogonia (SpG) were isolated from adult mouse testes. Cells were collected and counted prior to CUT&Tag library preparation. Following tagmentation and reverse crosslinking, DNA was purified using AMPure XP beads (Beckman Coulter, A63880) according to the manufacturer's instructions, and eluted in 22 µl Tris-HCl (pH 8.5). CUT&Tag libraries were prepared as described by Henikoff et al. (2020) (DOI: 10.17504/protocols.io.x54v9mkmzg3e/v5). Briefly, ~80,000 cells were bound to Concanavalin A-coated magnetic beads and incubated sequentially with primary and secondary antibodies, followed by pAG-Tn5 tagmentation at 55 °C for 1 h. DNA was released with SDS–Proteinase K treatment, PCR-amplified using NEBNext 2× PCR master mix (11 cycles, 98 °C 10 s / 60 °C 10 s), and purified with AMPure XP beads. Final libraries were quantified and assessed for quality before sequencing.