Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: For ChIPseq experiment, cells were treated as follows: 10x106 FBs were pelleted (either in the Unstimulated or TGFb-treated conditions) and suspended in 10ml DMEM and cross-linked in 1% formalin solution (Thermo Fisher Scientific) by rocking in room temperature for 10 minutes. Then glycine (final concentration 0.125M) was added to quench the cross-link for 5minutes. Samples were centrifuged at 1000 rcf for 5minutes at 4C. Cells were washed with 10ml of cold 1x PBS supplemented with proteinase inhibitors and phosphatase inhibitors (Roche # 4693132001) and the pellets were snap frozen in liquid nitrogen. All samples were stored at -80C until use. When ready, cell pellets were incubated in cell lysis buffer (20 mM Tris-HCl, pH 8, 85 mM KCl, 0.5% NP-40, protease inhibitors for 10 min on a rotator at 4C. Nuclei were isolated by centrifugation (2,500 x g, 5 min, 4C), resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8, 1% SDS, protease inhibitors) and incubated on a rotator for 30 min at 4C. Chromatin was sheared using a Covaris S2 sonicator (Covaris Inc) for 20 min (60 s cycles, 20% duty cycle, 200 cycles/burst, intensity = 5) until DNA was in the 200–700 base-pair range. Chromatin was diluted 3-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mMEDTA, 16.7mMTris-HCl, pH 8, 167 mM NaCl, protease inhibitors) and incubated with 2ul of anti-H3K27ac antibody (Abcam #ab4729) at 4C overnight under rotation. Antibody-protein complexes were immunoprecipitated using Pierce Protein A/G magnetic beads at 4C for 2 h under rotation. Beads were washed five times (2-min/wash under rotation) with cold RIPA buffer (50 mM HEPES- KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate), followed by one wash in cold final wash buffer (1xTE, 50 mM NaCl). Immunoprecipitated chromatin was eluted at 65C with agitation for 30 min in elution buffer (50mMTris-HCl pH 8.0, 10mMEDTA, 1% SDS). High-salt buffer (250mM Tris-HCl, pH 7.5, 32.5 mM EDTA, pH 8, 1.25M NaCl) and Proteinase K (New England Biolabs Inc (NEB)) were added and crosslinks were reversed overnight at 65C. Samples were treated with RNase A, and DNA was purified with AMPure XP beads (Beckman Coulter cat #A63881). Fragmented ChIP and input DNA were end-repaired, 5'-phosphorylated and dA-tailed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645). Samples were ligated to adaptor oligos for multiplex sequencing (NEB, E7335), PCR amplified, and sequenced on an Illumina NextSeq 500 at the Gladstone Institutes.