title: GSM6245675: ChIP-seq XEN cells, XistpA, H3K27ac, rep2; Mus musculus; ChIP-Seq
library_construction_protocol: STAR ChIP-seq was performed as previously described (Zhang et al., 2021; Zhang et al., 2016). Briefly, cells were first lysed and then subjected to MNase digestion by incubating with MNase working buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 and 0.2% Triton X-100) containing diluted MNase (Sigma, #N3755-200UN) at 37 °C for exact 5 min. The reaction was terminated by stop buffer (110 mM Tris-HCl pH 8.0 and 55 mM EDTA). After adding cold 2× RIPA buffer (1.8% Triton X-100, 280 mM NaCl, 0.2% SDS, 0.2% sodium deoxycholate (DOC), 5 mM EGTA and proteinase inhibitor) and spinning at max speed in 4 °C for 10 min, the soluble chromatin containing supernatant was incubated with 1mg H3K27ac antibody (ActiveMotif, #39133) in RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC, 1 mM EDTA and proteinase inhibitor) at 4°C overnight with rotation. Protein A/G dynabeads (Thermo Fisher Scientific) were used to bind the antibody at 4°C for 2 to 3 hours with rotation. The beads were then washed five times with RIPA buffer and once with LiCl buffer (10 mM Tris-HCl pH 8.0, 250mM LiCl, 1 mM EDTA, 0.5% NP-40 and 0.5% DOC). Beads bound chromatin were then digested with proteinase K (NEB, #P8107S) at 55°C for 90 min, followed by incubation at 72°C for 40 min to inactivate the proteinase K. STAR ChIP-seq libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, #E7645S)  following manufacturer's protocols. The libraries were purified and size-elected with AMPure XP beads (Beckman Coulter, #A63881).