ChIP-Seq: Cells were fixed for 15 min in 1% formaldehyde and quenched with 150mM glycine. Cells were washed 2x with cold PBS then re-suspended in 600µl lysis buffer (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 1x Roche Complete Protease Inhibitors), broken with 500µl zirconium beads  in an MP Biomedical Fast Prep (6 cycles 30s), then the lysate was separated from the beads and diluted to 1ml final volume in lysis buffer. Samples were sonicated 19x 30s in a Diagenode Bioruptor on High and cleared by centrifugation at 20,000g, 4°C for 5 min. 1µl anti-γH2A (Millipore 07-745-I) was added to 100µl lysate and incubated overnight at 4°C before addition of 15µl Gammabind beads (GE) in 25µl lysis buffer (pre-blocked with 1% BSA) and incubation for 2 hr at 4°C. Beads were washed 5 min each with lysis buffer, 0.5M salt lysis buffer, wash buffer (10mM Tris pH8.0, 0.25M LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA) and TE, then DNA eluted overnight at 65° in 200µl 50mM Tris pH8.0, 10mM EDTA, 1% SDS.  DNA was purified by phenol:chloroform extraction, ethanol precipitated and eluted in 50µl TE. mRNA-Seq: Total RNA was extracted using a mirVANA kit (Thermo Fisher) according to manufacturer’s instructions, with glass bead lysis as described in Cruz and Houseley 2014 ChIP-Seq: Indexed ChIP-Seq libraries were generated from 5ng ChIP/input with the NEBNext DNA Ultra kit (NEB) using NEBNext Multiplex Oligos for Illumina indexes (NEB). mRNA-Seq: Indexed mRNA-Seq libraries were constructed from 500ng total RNA using the NEBNext Ultra Directional RNA Library Prep Kit (NEB), with poly(A) selection using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB)
GEO Accession
GSM2299582