Protein G magnetic beads (NEB S1430S) were prepared by washing 3x in PBS with 0.05% bovine serum albumin (BSA), bound overnight to rabbit anti-H3K4me3 (Abcam, ab1342), rabbit anti-H3K27me3 (Millipore, 07-449), or mouse anti-Pol2 (Millipore, 17-672) at 2 µg/20 µl beads, and washed 3x in PBS-BSA. Material for immunoprecipitation (IP) as diluted 1:10 in dilution buffer (1% Triton X-100, 150mM NaCl, 2mM EDTA pH 8.0, 20mM Tris-HCl pH 8.0, protease inhibitors), and 20 µl of Protein G magnetic beads were added to each ml of sheared chromatin (about 200 µg each) and allowed to rotate overnight at 4° C. The following day, samples were washed 1x in low-salt wash (1% Triton X-100, 0.1% SDS, 150mM NaCl, 2 mM EDTA pH 8.0, 10mM Tris HCl pH 8.0, protease inhibitors), 2x in high-salt wash (1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA pH 8.0, 10mM Tris HCl pH 8.0, protease inhibitors), 2x in lithium wash (100 mM Tris HCl pH 9.0, 500mM LiCl, 1% NP40, 1% deoxycholic acid), 3x in TE (10mM Tris-HCl pH 8.0, 1mM EDTA), and eluted in 100 µl of elution buffer containing RNaseA and Proteinase K (1% SDS, 100 mM NaHCO3, 10 mg/ml RNaseA, 5 M NaCl, 0.2 mg/ml Proteinase K). Elutants and respective input controls were de-crosslinked overnight, at 65° C, purified on Qiagen Miniprep columns and eluted in ddH2O. One set from each condition was reserved for qPCR validation and the remaining ChIP DNA were speed-vac dried. DNA was resuspended in 15 µl of ddH2O, quantified with the Qubit fluorometer (Invitrogen) or the NanoDrop spectrophotometer (Thermo Scientific), until at least 10 ng were collected for each sample.  Single-end libraries were generated using the Illumina TruSeq ChIP seq library prep kit using 18 cycles for PCR sample enrichment, with the only protocol modification being size selection after fragment amplification. The library size selected was 200–300 bp in length and, after gel purification, size was confirmed with the Bioanalyzer (Agilent). Libraries were then sequenced on a 51 bp single-end run on the Illumina HiSeq 2000
GEO Accession
GSM1827869