GSM5742143
Cells were fixed using 2mM Di(N-succinimidyl) glutarate (DSG; Sigma-Aldrich, 80424-50MG-F) for 40 minutes and 1% methanol-free formaldehyde (Thermo Scientific, 28906) for 10 minutes in 1xPBS at room temperature, followed by 5 min blocking in 0.125 M glycine. Cells were washed twice with ice-cold PBS. The cell pellet was resuspended in Farnham buffer (5 mM PIPES, pH 8; 85 mM KCl; 0.5% Igepal). Cell suspensions were sonicated for 3 minutes in 1 ml Covaris tubes (Covaris, 520130) using Covaris S220 with the following settings: peak power = 75; duty factor = 2; cycles/ burst = 200. Isolated nuclei were washed with Farnham buffer and suspended in shearing buffer (10 mM Tris-HCl, pH 8; 0.1% SDS; 1 mM EDTA). Chromatin was sheared by sonication in 1-ml Covaris tubes using the following settings: peak power = 140; duty factor = 5; cycles/burst = 200, time = 25–30 min. Debris was removed by centrifugation at 16000 x g. A DNA fragment–size distribution of 200–600 bp was considered as ideal chromatin for ChIP. Chromatin was diluted 1:1 with IP buffer (10 mM Tris-HCl, pH 8; 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxicholate, 0.1% N-lauroylsarcosine) to achieve a final 0.05% SDS concentration. Good quality chromatin (200 μg) was used for immunoprecipitation. Protein G magnetic beads (Life Technologies, 10002D and 10004D, respectively) were incubated (rotated) with 5–10 μ g of FLAG antibody (Sigma-Aldrich, F1804-200UG) for 6 h at 4 °C. This bead–antibody complex was then incubated overnight at 4 °C with sheared chromatin. An aliquot of chromatin was saved as input DNA. Beads were washed and DNA–protein complexes were eluted from the beads by heating at 65 °C in elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS). Crosslinking was reversed for 6 h at 70 °C and samples were treated with 200 μg/ml RNase A (Thermo Scientific, EN0531) and 200 μg/ml proteinaseK (Sigma-Aldrich, P2308). ChIP DNA was purified with phenol–chloroform extraction and ethanol precipitation and used either for library preparation for next-generation sequencing. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep kit for Illumina (NEB E7645S). Some 2–5 ng of immunoprecipitated DNA was used for library preparation. Library size distribution was monitored by capillary electrophoresis (Agilent 2100 Bioanalyzer, High Sensitivity DNA Chips (Agilent, 5067–4626)). Libraries were sequenced paired-end on NextSeq500 or NovaSeq6000 instruments (Illumina). Libraries were prepared according to NEBNext Ultra II DNA Library preparation kit (NEB #E7645S).﻿Libraries were sequenced paired-end on HiSeq 2500, HiSeq 3000 or NextSeq 500 instruments (Illumina)