GSM3770502: NS_CHIP_INPUT_SF_Tbx5; Mus musculus; ChIP-Seq
Cells were crosslinked in 1% formaldehyde in suspension at room temperature for 10 minutes with gentle rotation. Crosslinking was quenched by addition of glycine (final 125 mM), followed by incubation at room temperature for 5 minutes with gentle rotation. 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 minutes on a rotator at 4°C. Nuclei were isolated by centrifugation (2,500 x g, 5 minutes, 4°C), 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 minutes at 4°C. Chromatin was sheared using a Covaris S2 sonicator for 15 minutes (60-second cycles, 5% duty cycle, 200 cycles/burst, intensity = 5) until DNA was in the 200–700 base-pair range. Chromatin was diluted five-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, protease inhibitors) and incubated with antibody (2 mg/million cells) at 4°C overnight under rotation. Antibodies used are Santa Cruz, Gata4, sc-1237x; Cell Signal Tech, Mef2c 5030; Santa Cruz, Tbx5 (C-20) sc-17866x. Antibody-protein complexes were immunoprecipitated using Pierce Protein A/G magnetic beads at 4°C for 2 hours under rotation. Beads were washed five times (2-minute washes 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 65°C with agitation for 30 minutes in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). High-salt buffer (250 mM Tris-HCl, pH 7.5, 32.5 mM EDTA, pH 8, 1.25 M NaCl) and Proteinase K were added and crosslinking was reversed by overnight incubation at 65°C. Samples were treated with RNase A and DNA was purified with Agencourt AMPure XP beads. Fragmented ChIP and input (whole-cell extract) DNA was 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.