[H3K27ac and RNA Polymerase II protocol] 3-6 x 10^7 crosslinked cells from each treatment group were resuspended in sonication buffer (0.5% SDS, 20mM Tris pH8.0, 0.5 mM EGTA, 2mM EDTA and protease inhibitor tablets (Roche)) in a ratio of 100ul cold sonication buffer to 1x10^7 cells. Cells were lysed on ice for 10min. Cell lysates were sonicated in a Qsonica at 4C. After a hard spin for 10 min at 4C, the supernatants corresponding to 2.5 million cells were removed and either flash frozen and kept at -80 (as input) or diluted into 1ml of ChIP buffer (0.5% Triton X-100, 2mM EDTA, 20mM TRIS pH8.0, 150mM NaCl, 10% glycerol) to be used for chromatin IP after a hard spin for 10min at 4C. The IP solution was incubated with selected IP antibody at 4C overnight tumbling. The next day proteinA dynabeads (Life Technologies) were added and after a 2hr incubation while tumbling at 4C, the IP samples were washed once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH8, 150mM NaCl), three times with high salt wash buffer (same as previous buffer but with 500mM NaCl), and once with LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% NADeoxycholate, 1mM EDTA, 10mM Tris pH 8). The beads were rinsed with TE buffer and the proteins were eluted with 500ul elution buffer (1%SDS, 0.1M NaHCO3) for 30 min at room temperature. At this point the input samples were also taken up in elution buffer.
GEO Accession
GSM2521744