GSM6257284: HEK293 PolII shCtrl ChIP-Seq; Homo sapiens; ChIP-Seq
5x10^6 cells were fixed in 1% PFA for 5 minutes and subsequently quenched in 125 mM glycine. Nuclear fraction was isolated and resuspended in sonication buffer (0.1% SDS, 50 mM TrisHCl, pH 8.0, 0.2 mM EDTA, 1x Protease inhibitor cocktail (Sigma)). Chromatin was sheared into 500 bp DNA fragments using Covaris AFA™ (Adaptive Focused Acoustics, Woburn, MA, USA) technology according to the manufacturer's instructions. After shearing, NaCl and NP40 were added to final concentration of 150 mM and 1%, respectively. Aliquots of extracts were precipitated by pre-coated Protein G Agarose-PLUS (Santa Cruz Biotechnology Inc.) with anti- RNA polymerase II (Abcam), THOC5 (Bethyl Laboratories. Inc.), CDK12 (Cell signaling), FLAG M2 antibodies or control IgG. Following 4 h rotation at 4°C, the beads were washed three times in RIPA buffer (150 mM NaCl, 1% NP40, 0.1 % SDS, 50 mM TrisHCl at pH 8), and two times in wash buffer (500 mM NaCl, 1% NP40, 0.1 % SDS, 100 mM TrisHCl at pH 8). Cross-links were reversed for 8 h at 65°C (250 mM NaCl) in the presence of RNAse A. Following proteinase K digestion at 55°C for 1 hour, the bound DNA fraction was isolated using NucleoSpin Extract II (Macherey-Nagel, Dueren, Germany). 5 ng of ChIP derived DNA were subjected to library preparation using TruSeq ChIP Library Preparation Kit (Illumia). Libraries were prepared and indexed according to manufacturer's protocol. Indexed libraries were pooled and sequenced on an Illumina HiSeq 2500.