title: Transcription factor NFYA directs male meiotic entry by regulating accessible chromatin at meiotic promoters in mice
design: Transcription factor NFYA directs male meiotic entry by regulating accessible chromatin at meiotic promoters in mice
library_name: CUT_Tag_CKO_rep3_p
library_construction_protocol: 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.