title: GSM5230806: H3K9me3_Pcgf6_WT_nESC; Mus musculus; ChIP-Seq
library_construction_protocol: EpiLC induction was performed essentially as described previously (Hayashi et al., Cell, 2011) with minor modifications. Briefly, pESCs were adapted to 2i (PD0325901, CHIR99021) + LIF feeder-free culture conditions to transit to nESCs. The EpiLCs were induced by plating nESCs in a well coated with human plasma fibronectin (Millipore) in N2B27 medium containing Activin A (R&D), bFGF (Gibco) and KnockOut Serum Replacement (KSR; Gibco). For cKO cells, 4-hydroxytamoxifen (4OHT; Sigma, dissolved in ethanol) at a final concentration of 100 nM was added to culture medium to induce Cre recombinase activity. ULI-N-ChIP-seq protocol (Brind'Amour et al., Nat Commun, 2015) was used for generating ChIP-seq libraries with some modifications. Briefly, chromatin was fragmentated by MNase (NEB), diluted in native ChIP buffer (20 mM Tris-HCl pH 8.0; 2 mM EDTA; 150 mM NaCl; 0.1% Triton X-100) containing 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche) and sonicated 5 second sonication cycles for 3 times with low power (Diagenode Bioruptor). Following pre-clearing of chromatin fragments with protein A and G beads (ThermoFisher Scientific), chromatin fragments were split and incubated with each of the histone methylation antibodies at 4 degree for overnight. Following purification of ChIPed DNA by AMPure XP beads, end repair and A-tailing were performed using NEBNext UltraII End Repair/dA-Tailing Module (NEB) and ligation was carried out using NEBNext Ultra II Ligation Module (NEB) with an in-house adaptor which is compatible with Illumina sequencers. After purification of adaptor-ligated products by AMPure XP beads, libraries were amplified with unique indexing primers for 14 cycles. Following purification of the amplified libraries by AMPure XP beads, libraries were pooled, run on 2% E-Gel EX Gel (ThermoFisher Scientific) and the fragments from 200-600 bp was excised and purified by Zymoclean Gel DNA Recovery Kit (Zymo Research).