GSM3934135: 2549-STC1_E; Homo sapiens; ChIP-Seq
ChIP assays were performed as described previously in Terranova et al (An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues; JoVE 2018). Briefly, 50mg of tissue or 20million cells were harvested via cross-linking with 1% (wt/ vol) formaldehyde for 10 min at 37 °C with shaking. After quenching with 150 mM glycine for 10 min at 37 °C with shaking, cells were washed twice with ice-cold PBS and frozen at −80 °C for further processing. Cross-linked pellets were thawed and lysed on ice for 30 min in ChIP harvest buffer (12 mM Tris-Cl, 1 × PBS, 6 mM EDTA, 0.5% SDS) with protease inhibitors (Sigma). Lysed cells were sonicated with a Bioruptor (Diagenode) to obtain chromatin fragments (~200–500 bp) and centrifuged at 15,000 × g for 15 min to obtain a soluble chromatin fraction. In parallel with cellular lysis and sonication, antibodies (5 μg/3 × 106 cells) were coupled with 30 μl of magnetic protein G beads in binding/blocking buffer (PBS + 0.1% Tween + 0.2% BSA) for 2 h at 4 °C with rotation. Soluble chromatin was diluted five times using ChIP dilution buffer (10 mM Tris-Cl, 140 mM NaCl, 0.1% DOC, 1% Triton X, 1 mM EDTA) with protease inhibitors and added to the antibody-coupled beads with rotation at 4 °C overnight. After washing, samples were treated with elution buffer (10 mM Tris-Cl, pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS), RNase A, and Proteinase K, and cross-links were reversed overnight at 37. Immune complexes were then washed five times with cold RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), twice with cold high-salt RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 500mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), and twice with cold LiCl buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC).  ChIP DNA was purified using AMPure XP beads (Agencourt) and quantified using the Qubit 2000 (Invitrogen) and Bioanalyzer 1000 (Agilent). Libraries for Illumina sequencing were generated following the New England BioLabs (NEB) Next Ultra DNA Library Prep Kit protocol. A total of 10 cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments (~200–500 bp) and quantified using the Qubit 2000 and Bioanalyzer 1000 before multiplexing. Libraries for Illumina sequencing were generated following the New England BioLabs NEBNext Ultra DNA Library Prep Kit protocol. A total of 10x cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments ~200 to 500bp and quantified using the Qubit 2000 and TapeStation 2000 before multiplexing.