title: GSM4829306: glyr1Ab7_ipsc.2 ChIPseq rep 2; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIPseq experiments, CPs (30×106 for cTFs and 10×106 for GLYR1 and histone marks) were pelleted and suspended in 10ml DMEM and cross-linked in 1% Formaldehyde solution (Cat. 28906, Thermo Fisher Scientific) by rocking in room temperature for 10 min. Then glycine (final concentration 0.125M) was added to quench the cross-link for 5 min. Samples were centrifuged at 1000 rcf for 5 min. at 4°C. Cells were washed with 10ml of cold 1x PBS supplemented with proteinase inhibitors and phosphatase inhibitors (Cat. 4693132001 Roche and Cat. 4906837001 Sigma-Aldrich) and the pellets were snap frozen in liquid nitrogen. All samples were stored at −80°C 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/phosphatase inhibitors) for 10 min. on a rotator at 4°C. Nuclei were isolated by centrifugation (2,500 x g, 5 min., 4°C), resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8, 1% SDS, protease/phosphatase inhibitors) and incubated on a rotator for 30 min. at 4°C. Chromatin was sheared using a Covaris S2 sonicator (Covaris Inc) for 15 min. (60 s cycles, 5% duty cycle, 200 cycles/burst, intensity = 6) 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/phosphatase inhibitors) and incubated with the corresponding primary antibody at 4°C overnight under rotation. Antibody-protein complexes were immunoprecipitated using 50μl of Dynabeads™ Protein A/Protein G (Cat. 10015D, Invitrogen) per sample at 4°C for 2 h under rotation. After incubation, 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 65°C 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 (Cat. P8107s, New England Biolabs Inc (NEB)) were added and crosslinks were reversed overnight at 65°C. Samples were treated with RNase A, and DNA was purified with AMPure XP beads (Cat. A63881, Beckman Coulter). Primary antibodies used for ChIP were: GATA4 (Cat. sc-1237 X, Santa Cruz), TBX5 (Cat. sc-17866 X, Santa Cruz), H3K36me3 (Cat. ab9050, Abcam), GLYR1 (anti-serum 7; provided by James T. Kadonaga's laboratory), NKX2-5 (Cat. sc-8697 X, Santa Cruz), MEIS1 (Cat. ab19867, Abcam). Fragmented ChIP and input DNA were end-repaired, 5'-phosphorylated and dA-tailed with NEBNext Ultra II DNA Library Prep Kit for Illumina (Cat. E7645, New England BioLabs). Samples were ligated to adaptor oligos for multiplex sequencing (Cat. E7335, New England BioLabs), PCR amplified, and sequenced on an Illumina NextSeq 500 at the Gladstone Institutes.