Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: cells were cross-linked with 1% formaldehyde for 10 min, followed by quenching with 125 mM glycine for 5 min at room temperature. Fixed cells were washed with TBS and the pellets were kept on -70 °C. The pellets were thawed and resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) once and incubated for 20 minutes at 37 °C in the presence of 1,000 Gel units of MNase (NEB, M0247S) in 250 ul reaction volume. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% Sodium deoxycholate), the lysates were sonicated for 5 min (30 sec-on / 30 sec-off) in Diagenode bioruptor and centrifuged at 15,000 rpm for 10 min. ChIP performed with rabbit polyclonal anti-BRD4 (Bethyl Laboratories, catalog# A301-985A). Ovation Ultralow DR Multiplex system (NuGEN).