GSM4307714: A549, normoxia, replicate 1, HIF1A ChIP; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation. Sub-confluent cultures of each cell line (HCT116, RKO, A549, and H460) were placed in normoxic or hypoxic conditions for 24 hours. After treatment, media was removed and replaced with 1% formaldehyde in PBS (equilibrated by bubbling for 20 min with a mix of 1% O2, 5% CO2, and 94% N2 for hypoxia samples). To avoid rapid degradation of HIF1A, a crosslinking time of 5 min was used for all samples and terminated by addition of glycine to 0.125 M final concentration. Following 5 min of formaldehyde quenching, plates were placed on ice and washed three times with ice-cold PBS. Subsequently, cells were lysed in RIPA buffer and sonicated to generate DNA fragments of ~200-300 bp (Qsonica Q800R, 70% amplitude, 30 sec on/30 sec off cycle, 20 cycles for H460 lysates, 25 cycles for HCT116 and RKO lysates, and 30 cycles for A549 lysates). Samples were centrifuged at 20000 g for 20 min at 4°C and protein concentration in collected supernatants was measured with a BCA Protein Assay Kit. At this step, input samples (50 µg of total protein per cell line) were set aside. For each sample, four 1 ml aliquots each containing 1 mg of total protein were used for immunoprecipitation. Samples were pre-cleared with 15 µl of RIPA-washed Dynabeads Protein G (Invitrogen, Thermo Fisher Scientific) by rocking for 1 hour at 4°C. The supernatant was then collected and incubated with 30 µl of Dynabeads and 5 µl of anti-HIF1A antibody (Novus Biologicals, NB100-134) incubated overnight on a rocker at 4°C. Next, beads were washed twice with RIPA buffer, twice with IP wash buffer (500 mM LiCl, 100 mM Tris pH 8.5, 1% v/v NP-40, 1% w/v sodium deoxycholate), and twice with RIPA (2 min on rocker at 4°C for each washing step). Immunocomplexes were eluted by resuspending beads in 100 µl TE buffer and 200 µl of elution buffer (70 mM Tris pH 8, 1 mM EDTA and 1.5% w/v SDS) and incubating for 10 min at 65°C. Both eluted immunocomplexes and input samples were combined with NaCl solution to a final concentration of 200 mM and incubated overnight at 65°C to reverse formaldehyde crosslinks, followed by treatment with 20 µg proteinase K. DNA fragments recovered using phenol/chloroform extraction followed by ethanol precipitation and re-dissolved in TE buffer. Library preparation. DNA fragments were size-selected (80-600 bp) by agarose gel electrophoresis (2% gel, BluePippin) and used for barcoded library preparation with the NEBNext Ultra II DNA kit, according to the manufacturer's instructions (New England Biolabs). Final libraries were size-selected (200-600 bp, BluePippin) and analyzed on Bioanalyzer High Sensitivity DNA chips (Agilent) to confirm fragments ranging from 200 to 400 bp. Single-end 150 bp sequencing of pooled barcoded libraries was carried out on the Illumina HiSeq 4000 platform by the Genomics Core facility at the University of Colorado Anschutz.