title: GSM6737699: HUWE1-WT, shCtrl, ChIP WRNIP1, rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: For each ChIP sample, 4 - 6 x107 HCT116 cells per condition were fixed with 1% PFA for 4 min at room temperature and the reaction was quenched by adding 200 mM glycine for 4 min. Cells were scraped in ice cold PBS and lysed in 200 µl PBS, 0.5% (v/v) TritonX-100, 1 mM dithiothreitol, 5 mM MgCl2, 5 mM CaCl2 and protease/phosphatase inhibitors (Sigma) for 10 min on ice. Chromatin was fragmented with 1 µl micrococcal nuclease (NEB M0247S) for 3 min at 37°C and 20 µl 500 mM EDTA was added to suppress enzyme activity. After diluting samples with 800 µl TNT-300 (25 mM Tris, 300 mM NaCl, 1% (v/v) Triton-X100, pH 7.4, with protease/phosphatase inhibitor cocktails), chromatin was further fragmented by sonication for 3x 2 min with 30% amplitude, 2 W power and a 45 s on / 15 s off cycle with an UP200St sonicator (Hielscher) on ice. Lysates were cleared by centrifugation for 10 min at 17,000xg and 2% of each sample were separated as input. For each immunoprecipitation 1.5 - 2 µg of target specific antibody was added to the lysate and rotated overnight at 4°C. To capture proteins of interest, 10 µl magnetic protein A or protein G bead slurry (Thermo) were added per sample and rotated for 2 h. Beads were washed three times with TNT-300, 0.1% SDS and RNA was digested with 1 µl RNAseA (Thermo) in 10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS, 250 mM NaCl for 1 h at 50°C. Proteins were digested with 2 µl proteinase K (Thermo) at 50°C for 1 h followed by 65°C overnight. Fragmented DNA was purified using phenol/chloroform/isoamyl (Roth A156.1) Sequencing libraries were prepared using NEB Ultra II DNA library Prep kit according to the manufacturer's instructions.