title: GSM5683117: ChIPseq_RNAPolymeraseII_OPM2_2hours_DMSO; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were washed once in ice-cold PBS prior to crosslinking. For crosslinking, 1/10th volume of fresh formaldehyde solution (11% formaldehyde, 0.05mM EGTA, 1mM EDTA, 100mM NaCl, 50mM Hepes-KOH pH 7.5) was added and incubated for 20 minutes at room temperature with rotation. Crosslinking was quenched by addition of 1/20th volume of 2.5M glycine and incubated for 5 minutes at room temperature with rotation. For isolation of nuclei, cell pellets were washed once in ice-cold PBS and then resuspended in ice-cold nuclear extraction buffer (0.5% NP-40, 2mM EDTA, 10mM NaCl, 20mM Tris-HCl pH 8) and incubated for 5 minutes on ice. Following three sequential incubations in nuclear extraction buffer, cell nuclei were pelleted and resuspended in sonication buffer (0.3% SDS, 1% NP-40, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 7.5) at a concentration equivalent to 50e6 cells per mL. Samples were sonicated in 12x24mm Covaris tubes using the Covaris S2 instrument for 18 minutes using the following settings: 20% Duty Cycle, 1000 cycles/burst, and 10 Intensity. Prior to immunoprecipitation, sheared chromatin was diluted 1:1 in ChIP dilution buffer (1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8) and quantified using Qubit dsDNA HS assay kit. For ChIP-Rx, sheared Drosophila chromatin from S2 cells was spiked into immunoprecipitations at 1:40 ratio of Drosophila:human and processed as a single sample until ChIP-Rx normalization following DNA sequencing. Immunoprecipitations were performed overnight (12-16 hours, 4°C, with rotation) using Protein A and Protein G Dyna beads (Invitrogen) and the indicated antibody. Samples were washed once with ChIP dilution buffer, wash buffer 1 (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8), wash buffer 2 (0.5% deoxycholate, 0.5% NP-40, 2mM EDTA, 250mM LiCl, 20mM Tris-HCl pH 8), and TE buffer (1mM EDTA, 10mM Tris-HCl pH 7.5) prior to incubation in reverse crosslinking buffer (200mM NaCl, 100mM NaHCO3, 1% SDS, 300μg/mL Proteinase-K) for 4 hours at 55°C with shaking. Finally, the supernatant was reverse-crosslinked overnight (12-16 hours) at 65°C prior to ChIP DNA isolation using Zymogen ChIP DNA Clean and Concentrator Kit (Zymo Research, D5205). Libraries were generated from ChIP DNA using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads.