GSM5707545: IP_HIF1A; Mus musculus; ChIP-Seq
DMOG-treated Myc-CaP cells were incubated in 1% paraformaldehyde for 8 min and quenched with 0.125 M glycine buffer for 5 min at room temperature. Cross-linked cells were resuspended in 500 µL of 10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% (v/v) NP40, 1 mM DTT, and 1 mM PMSF (pH 7.6) and incubated on ice for 5 min. After centrifugation at 400 g for 5 min, nuclear pellets were resuspended in ChIP lysis buffer (50 mM Tris-HCl pH 8, 0.1% SDS, 10 mM EDTA) and sonicated in 300–400 bp DNA fragments (Covaris). 50 µg of chromatin were immunoprecipitated in ChIP buffer (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) using 5 µg of HIF1A antibody (CST 36169) or a rabbit IgG as negative control. 30 µL of Dynabeads Protein G (ThermoFisher Scientific) were added to the immunocomplexes for 2 h at 4°C. DNA was recovered by reverse crosslinking overnight at 65°C, and extracted using phenol/chloroform/isoamyl alcohol. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 0 TO 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 + --- cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.