title: GSM2668157: S9.6 DRIP Seq_2; Homo sapiens; OTHER
library_construction_protocol: DRIP was performed as described in Ginno et al., 2012. Briefly, DNA was extracted with phenol/chloroform in phase lock tubes (5Prime), precipitated with EtOH/sodium acetate, washed with 70% EtOH, and resuspended in TE. DNA was digested with EcoRI, XbaI, SspI, BsrgI, and HindIII (NEB) restriction enzymes overnight at 37 °C. For RNase H-treated samples, 4 ug of DNA was treated with RNase H (NEB, M0297S) overnight at 37 °C. DNA was purified by phenol/chloroform, EtOH/sodium acetate precipitation as described above. 4μg of DNA was bound with 10 μg of S9.6 antibody in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4°C. Protein A/G sepharose beads (Pierce) were added for 2 h. Bound beads were washed 3 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55°C. DNA was purified as described. In addition, sheared Drosophila melanogaster chromatin (Active Motif, 53083) and a Drosophila-specific H2Av antibody (Active Motif, 61686) was spiked-in as a minor fraction of the DRIP DNA to allow normalization of read counts according to the manufacturer's protocol. Using asynchronously growing HeLa cells, DRIP followed by library preparation, next generation sequencing, and peak calling were performed as described in Stork et al., 2016 with minor modifications. RNase A pre-treatment before DRIP was conducted with 6 μg/ml of enzyme for 45 min at 37ºC in 10 mM Tris-HCl pH 7.5 supplemented with 0.5M NaCl as described in Sanz et al., 2016 . RNase A pre-treatment before DRIP was conducted with 6 μg/ml of enzyme for 45 min at 37ºC in 10 mM Tris-HCl pH 7.5 supplemented with 0.5M NaCl as described in Sanz et al., 2016 . Libraries were pooled and sequenced on an Illumina HiSeq 4000 machine with paired-end 75bp reads at the Stanford Functional Genomics Facility (NIH grant S10OD018220).
attributes.tag: GEO Accession
attributes.value: GSM2668157