title: GSM1910645: PARP-1KO_ESC_Oct4_ChIPseq_Rep1; Mus musculus; ChIP-Seq
library_construction_protocol: mESCs were passaged twice without a feeder layer and then grown in geletin-coated plates to ~70 to 80% confluence.  The cells were cross-linked using 1% paraformaldehyde at room temperature for 10 min., followed by quenching in 125 mM glycine for 5 min. at 4°C.  The crosslinked cells were collected, and the nuclei were released by gentle pipetting three times in lysis buffer [10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 0.5% NP-40, 10% glycerol, 1 mM DTT, 1x protease inhibitor cocktail (Roche)].  The nuclei were collected by gentle centrifugation and resuspended in sonication buffer [1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor cocktail (Roche)].  The nuclei were then incubated in sonication buffer on ice for 10 min., and sonicated at 4°C using a Biorupter (Diagenode) high setting, three cycles of 5 min. sonication (30 seconds on/30 seconds off) with 5 min. intervals) to generate genomic DNA fragments of 200 - 500 bp in length.  The sonicated chromatin was clarified by centrifugation and pre-cleared with agarose beads. Aliquots of the pre-cleared chromatin were immunoprecipitated with various antibodies at 4°C overnight, followed by collection of the immunoprecipitates using protein A (Millipore) or G (Invitrogen) agarose beads (protein A beads for H3K4me3, H3K27me3, and PARP-1 antibodies; protein G beads for Sox2 and Oct4 antibodies).  The beads were collected by gentle centrifugation and washed in low salt wash buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x protease inhibitor cocktail), high salt wash buffer (low salt wash buffer containing 500 mM NaCl), and LiCl wash buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1x protease inhibitor cocktail).  The immunoprecipitated genomic DNA was eluted and the crosslinks were reversed by incubation in elution buffer (100 mM NaHCO3, 1% SDS) at 65°C overnight.  The genomic DNA was then deproteinized by digestion with proteinase K and extraction with phenol:chloroform:isoamyl alcohol, followed by precipitation with ethanol. Approximately 50 ng of ChIPed DNA (quantified using a NanoDrop) was used to prepare each ChIP-seq library. The genomic DNA fragments were end-polished, dA-tailed, and ligated to Y-adaptors containing barcode sequences.  After agarose gel-based size selection and purification, the DNA was amplified for 13 - 15 cycles by PCR using Phusion high-fidelity DNA polymerase (NEB).  The final ChIP-seq libraries were subjected to quality control assessment using a Bioanalyzer (Agilent), followed by 50 bp sequencing using an Illumina HiSeq 2000 Sequencing System.