ChIP and qChIP

MCF-7 or T47D cells treated with hypoxia (1% O₂) for 24 h were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched by the addition of glycine to a final concentration of 125 mM for 5 min. The fixed cells were resuspended in lysis buffer (1% SDS, 5 mM EDTA, and 50 mM Tris–HCl, pH 8.1) containing protease inhibitors, then subjected to 30 cycles (30 s on and off) of sonication (Bioruptor, Diagenode) to generate chromatin fragments of ∼300 bp in length. Lysates were diluted in buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris–HCl, pH 8.1) containing protease inhibitors. For immunoprecipitation, the diluted chromatin was incubated with normal IgG (control) or HIF-1α antibodies overnight at 4°C with constant rotation, followed by incubation with 50 μl of 50% (v/v) protein A/G Sepharose beads for an additional 2 h. Beads were successively washed with the following buffers: TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris–HCl, pH 8.0); TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris–HCl, pH 8.0); TSE III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris–HCl, pH 8.0). The pulled-down chromatin complex eluted by TE (1 mM EDTA and 10 mM Tris–HCl, pH 8.0) and input were de-crosslinked at 55°C for 12 h in elution buffer (1% SDS and 0.1 M NaHCO₃) (Li et al., 2016). The DNA was purified with the QIAquick PCR Purification Kit (QIAGEN). qChIPs were performed using Power SYBR Green PCR Master Mix and an ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, United States). The primers used were: JFK: 5′-TGAGCACATTTGTGTGCCG-3′ (forward) and 5′-TGGACACTACTAGGCGGTCA-3′ (reverse).