GSM1634924: HeLa E2F7_hypoxia; Homo sapiens; ChIP-Seq
ChIP was performed according the EZ ChIP protocol (Upstate, 17-371) with the following specifications: HeLa cells were seeded at day 1 at a concentration of 7x10^6 cells per 145mm plate. Next day, cells were cultured overnight in hypoxia, or continued to grow under normoxia, and harvested on day 3. For single and double ChIP, 5 145mm plates were used per condition. In vitro crosslinking was performed on a shaker at room temperature (RT) for 10 minutes, using 1% freshly made paraformaldehyde. Crosslinking was quenched for 5 minutes incubation at RT on a shaker. Next, cells were washed twice on ice with PBS (40C, supplemented with protease inhibitors (11873580001, Roche)), and harvested, pelleted and resuspended in 2ml lysis buffer (0.3% SDS, 10mM EDTA, 50mM Tris and protease inhibitors). Sonication (10 cycles of 10 seconds followed by 1 minute incubation on ice) was performed in a FACS tubes using a Soniprep 150 sonicator (MSE). 10µl of sonicated sample was analyzed on gel to check for sonication efficiency. Sonicated lysates were centrifugated (10 minutes, 40C) to remove insoluble components and large DNA fragments. 200µL lysate was used per ChIP sample. The DNA concentration in the sonicated lysate was measure using a Nanodrop to normalize the amount of input DNA between normoxic and hypoxic ChIP samples. Protein G agarose beads (16-266, Milipore) were coated overnight in 0,1% BSA (Sigma, A3294), and 60 µL was used for pre-clear (2h, 40C) and final precipitation of immune complexes (1h, 40C). For single ChIP 5µg and for double ChIP 10µg was used. immunorecipitation were performed overnight at 40C on a rotating platform. In case of double ChIP, eluates were diluted in dilution buffer and incubated with another 10ug of antibody overnight. The following antibodies were used: ChIP grade HIF1α (ab2185, Abcam) E2F7 (sc-66870), E2F1 (sc-193), IgG (2729S; cell signaling). De-crosslinked DNA was purified over a column (Qiagen, 28106) and eluted in 65 µl H2O immunoprecipitated chromatin was sheared, end-repaired, sequencing adaptors were ligated and the library was amplified by ligation mediated PCR (LMPCR). After LMPCR, the library was purified and checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel, barcoded and sequenced on SOLiD/AB sequencer in multiplexed way to produce 50-bp long reads.
GEO Accession
GSM1634924