Instrument: Illumina Genome Analyzer II
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were incubated in lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.2 % NP-40) for 1 hour at 4 ˚C. Chromatin was digested at a concentration of 25 x106 cells/mL with 350 U/mL MNase (Roche,10107921001) for 20 minutes at RT in MNase reaction buffer (55 mM Tris-HCl pH 7.5, 0.05 mM EDTA, 2.5 mM MgAc2, 12.5 % glycerol, 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2). The digestion was stopped with 10 mM EDTA. The chromatin was then sonicated with a 550 Sonic Dismembrator (Fisher Scientific) on power 4, using cycles of 0.7 seconds on and 1.4 seconds off for a total processing time of 3 minutes. Lysates were centrifuged at 21,000 x g for 10 min at 4 ˚C. 5 x106 cell equivalents were used for each ChIP assay, diluted to 1 mL with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 1.1% Triton-X 100, 167 mM NaCl, 0.01 % SDS). Chromatin was pre-cleared for 2 hours at 4 ˚C with Protein A Dynabeads (Life Technologies,10001D) in blocking buffer (1x PBS, 0.5 % BSA, 0.1% Tween-20). Beads were captured and pre-cleared chromatin was incubated with antibodies overnight at 4 ˚C. Samples were then incubated with Protein A Dynabeads in blocking buffer for 2 hours at 4 ˚C. Beads were washed 3x with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS) and 1x with RIPA high salt buffer (10 mM Tris-HCl pH 8.0, 360 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS). Bound chromatin was eluted with elution buffer (10 mM Tris-HCl, 1 % SDS, 300 mM NaCl, 10 mM EDTA pH 8.0). Crosslinks were reversed by incubation at 65 ˚C for 6 hours. Eluted chromatin was treated with 150 U/mL RNase A for 30 minutes at 37 ˚C and digested with 75 µg/mL proteinase K for 1 hour at 55 ˚C. ChIP DNA was purified with using a MinElute PCR Purification Kit (QIAGEN) and quantified with a Qubit fluorimeter (Life Technologies). Library DNA fragments for BRD4, RNAPII, RNAPIISer2P ChIP and input DNA were ligated to Illumina adapters and size selected (200-250 bp) on a 2% agarose gel and PCR amplified for 18 cycles. Libraries of H3K27ac ChIP and input DNA were constructed using an Ovation Ultralow DR Multiplex System Kit (NuGEN) using 15 cycles of amplification. Libraries were size selected (250-300 bp) on a 2% agarose gel.