GSM2176717: 2hr S 9.6 DRIP Seq; Homo sapiens; OTHER
DRIP was performed as described in Ginno et al., 2012. Briefly, DNA was extracted carefully by phenol/chroloform extraction in phase lock tubes, precipitated with EtOH/sodium acetate, washed with 70% EtOH, and resuspended in TE. DNA was digested with a cocktail of restriction enzymes (Bsrg1, EcoR1, HindIII, SspI, XbaI) overnight at 37˚C. For RNase H-treated samples, 8 ug of DNA was treated with RNase H overnight at 37˚C. DNA was purified by standard methods described above. 4ug of DNA was bound with 10 ug 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 were added for 2 hours. 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. DNA libraries were prepared from 3 pooled DRIPs by sample. Briefly, input and DRIP DNA was sonicated to approximately 300 – 700 bp on a Bioruptor (Diagenode). End repair (NEB E6050S), A-tailing (NEB M0212S), ligation of adapters (NEB M2200S; Affymetrix Prep2Seq Adapters 79800), and PCR amplification were performed by standard methods as described (Ginno et al., 2012). Ampure XP beads (Beckman Coulter, A63880) were used for size selection. Libraries were pooled and sequenced on an Illumina HiSeq machine with single-end 50 bp reads by Elim BioPharm (Hayward, CA).
GEO Accession
GSM2176717