GSM5832706: Gibbin_hESC_rep2; Homo sapiens; ChIP-Seq
Briefly, cells were detached with Accutase and crosslinked in solution for 10 minutes in freshly prepared 1% formaldehyde (Thermo Scientific) in PBS. Cell were quenched in 0.125M glycine, followed by two washes with cold PBS. 25x106 cross-linked cells were used per ChIP replicate. Cells were lysed in lysis buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 0.5% SDS, 1X protease inhibitors) for 30 minutes on ice and sonicated twice for 10 minutes on a Diagenode Bioruptor to achieve a chromatin between 200 and 400bp in size. Chromatin was centrifuged, quantified and diluted in dilution buffer up to 2mL (50mM Tris-HCl pH 8.0, 10mM EDTA, 1X protease inhibitors). Sheared chromatin was incubated overnight at 4°C with a TFAP2A antibody. The next day lysates were incubated with 30μL of Dynabead protein G beads (Invitrogen) for 4 hours at 4°C. Beads were washed twice each with low salt buffer (50mM Tris-HCl pH 8.0, 0.15M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), high salt buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), and LiCl buffer (50mM Tris-HCl pH 8.0, 0.15M LiCl, 1mM EDTA pH 8.0, 1% Nonidet P-40, 0.1% sodium deoxycholate). DNA was eluted in 100μL of elution buffer (50mM NaHCO3, 1% SDS) and crosslinks were reversed with 4μL of 5M NaCl incubated shaking for 16 hours at 67°C. RNA was removed by adding 1μL of 10mg/mL RNase A for 30 minutes at 37°C. DNA was cleaned using the Qiaquick PCR purification kit (Qiagen) and quantified using Qubit (Invitrogen). 10ng of DNA was used to make libraries with the NEBNext kit (New England Biolabs) and AMPure XP beads (Beckman Coulter) according to the manufacturer instructions.