alias: GSM4544619
title: GSM4544619: MCF7-Vector _CDYL2 ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were grown to sub-confluence in 15 cm tissue culture treated plates (Corning), cross-linked, washed and pelleted. The resulting cell pellets were pre-treated by incubating in lysis buffer A (Lysis Buffer 1 (50 mM HEPES pH 7.5; 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) for 10 minutes at 4°C, then lysis buffer B (10 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1× protease Inhibitors) for 10 minutes at room temperature, as previously described (Lee et al., 2006). They were then lysed in buffer C (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1%; Na-Deoxycholate, 1× protease inhibitors) supplemented with 0.5% SDS, incubated on ice for 30 minutes with occasional vortexing, then sonicated on ice to an average fragment size of 150 bp using a sonicator (Branson, Digital Sonifier 450). The sonicated lysate was centrifuged at 12,000 r.p.m. in a benchtop centrifuge at 4°C, the supernatant diluted five times in buffer C, and 1 mL aliquots of this added to 50 µL of magnetic protein A beads (Invitrogen) pre-coated with 5 µg of anti-CDYL2 IgG (MyBiosource, MBS821304). These were incubated overnight at 4°C with rotation, then pelleted and washed with two sequential additions each of wash buffer 1 (0.1% SDS, 1%Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8, 150mM NaCl), wash buffer 2 (0.1% SDS, 1%Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8, 500mM NaCl), wash buffer 3 (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8), and TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0, 50 mM NaCl). Chromatin was eluted by incubating for 30 minutes at 65°C  in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS) with frequent vortexing. Crosslinks were reversed by overnight incubation at 65°C, and eluates treated with RNAse A (Sigma) for 2h, followed by Proteinase K for 2h, then extracted using a classical Phenol-Chloroform/Ethanol precipitation protocol. Pair end DNA sample libraries were prepared using the MicroPlex Library Preparation Kit v2 (Diagenode) according to the manufacturer's instructions.
identifiers.namespace: GEO
identifiers.id: GSM4544619