GSM2754209: FA_IgG_control; Homo sapiens; ChIP-Seq
Immediately after irradiation, OCI-Ly1 cell suspensions were added to ice cold PBS (pH 7.4) containing protease inhibitors according to the manufacturer’s recommendations (Complete Protease Inhibitor, Roche Applied Science). All subsequent steps were performed at 4°C. After centrifugation at 1,000 g for 7 min, the pellet was resuspended in 250 µl of ChIP lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, protease inhibitors). Chromatin was sheared on ice (Microtip sonicator, Labsonic U, Sartorius) into fragments averaging 250 bp and centrifuged at 16,000 g for 10 min. Fragmentation of chromatin was analyzed with purified DNA using agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent Technologies). An aliquot of sheared chromatin was used for UV input DNA sequencing. The supernatant was pre-cleared for 1 h using pre-washed protein A magnetic beads according to the manufacturer’s instructions (Dynabeads Protein A, Invitrogen). A ChIP-grade anti-Bcl6 antibody (Bcl-6 (N-3), sc-858, Lot# A3013, Santa Cruz) was used for ChIP. The specificity and suitability for ChIP was confirmed by immunoblot analysis. For UV IgG control ChIP, normal rabbit IgG (sc-2027, Lot# D1513, Santa Cruz) was used. Clarified chromatin extracts were incubated for 12 h with specific anti-Bcl6 antibody and control IgG antibody, respectively. Nucleoprotein-antibody complexes were precipitated using pre-washed beads for 1 h. The supernatant was removed and beads were sequentially washed twice in low salt (0.1% SDS, 1% Triton-X 100, 150 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA), high salt (0.1% SDS, 1% Triton-X 100, 500 mM NaCl, 20 mM Tris-HCl pH 8.0, 2 mM EDTA), LiCl (0.5% NP-40, 0.5% deoxycholic acid (DOC), 250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) and TE (10 mM Tris pH 8.0, 1 mM EDTA) buffer. For elution of immunoprecipitated nucleoprotein-antibody complexes, beads were resuspended in 0.1 M citrate buffer (pH 2.2) for 2 min at room temperature. The supernatant was removed and transferred into Tris-HCl (pH 8.0) for neutralization. After RNase A treatment (50 µg/ml, Fermentas) for 1 h at 37°C and Proteinase K treatment (100 µg/ml, Fermentas) for 12 h at 50°C, genomic DNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. Formaldehyde (FA) ChIP was performed as described except for the following steps. Cells were harvested, washed twice with ice cold PBS (pH 7.4) and lysed in RIPA buffer (1% NP-40, 0.5% DOC, 0.1% SDS, 150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA, protease inhibitors). For elution of immunocomplexes, beads were resuspended in elution buffer (1% SDS, 0.1 M NaHCO3) and crosslinking was reverted by addition of 0.3 M NaCl and incubation for 5 h at 65°C. Sequencing libraries were prepared from UV-ChIP, FA ChIP and control DNA fragments according to the manufacturer’s protocol (NEBNext® Ultra™ DNA Library Prep Kit). In brief, DNA fragments were end-repaired and the blunt, phosphorylated ends were treated with Klenow DNA polymerase and dATP to yield a 3’ A base overhang for ligation of adapters. After adapter ligation, DNA was PCR amplified (15 cycles). Libraries were size-selected to achieve ChIP DNA fragment lengths of 200-300 bp. The purified DNA was captured on an Illumina flow cell for cluster generation and libraries were sequenced on a HiSeq 2500 (Illumina) instrument according to manufacturer's protocol.
GEO Accession
GSM2754209