title: GSM9256409: NCC d5 NOG rep10; Homo sapiens; ChIP-Seq
library_construction_protocol: 10 million hiPSC-NCCs were crosslinked in 1% paraformaldehyde for 5 min at RT with mild rotation. Crosslinking was quenched with 0.125M glycine by rotation for 5 min at RT. Cells were washed twice with cold PBS and were snap-frozen for 15 min on dry ice. Samples were stored at -80 °C until further processing. Cells were resuspended in 1 mL ChIP buffer (150 mM NaCl, 1% Triton X-100, 500 mM DTT, 10 mM Tris-HCl, 5 mM EDTA) supplemented with Pierce protease inhibitor tablet and incubated on ice for 10 min. SDS was added to each sample to a final concentration of 0.3%, and chromatin was sheared using the following settings: 9 min, duty factor 5%, 6°C in a Covaris M220 Focused-Ultrasonicator. Chromatin fragment size was assessed on a Tapestation 2200. The chromatin lysates were then centrifuged at 13,000 g for 10 min at 4 °C and supernatant was subsequently incubated with ChIP buffer supplemented with Pierce protease inhibitor and Dynabeads Protein G magnetic beads (Invitrogen) along with 3 μg of H3K9me3 antibody (Abcam, ab8898) and incubated overnight at 4 °C under mild rotation. The following day, samples were placed in a magnetic rack and washed twice with Mixed Micelle Wash Buffer (150 mM NaCl, 1% Triton X-100, 0.2% SDS, 20 mM Tris-HCl, 5 mM EDTA, 65% sucrose), twice with Buffer 200 (200 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 25 mM HEPES, 10 mM Tris-HCl, 1 mM EDTA), twice with LiCl/detergent wash (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 10 mM Tris-HCl, 1 mM EDTA), once with cold TE. The beads were resuspended in TE + 1% SDS and incubated at 65 °C for 10 min at 1200 rpm to elute immunocomplexes. The elution was repeated twice and the samples were incubated overnight at 65˚C to reverse cross-linking. The following day, Proteinase K (0.5 mg/mL) was added to digest samples at 65 °C for 1h. DNA was purified using the Zymo ChIP DNA Clean and Concentrator kit (Zymo, D5205) and quantified with the Quantifluor ONE dsDNA system (Promega, E4871). Barcoded libraries were made with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) using NEBNext Multiplex Oligos Dual Index Primers for Illumina (NEB, E7600S), and sequenced on Illumina NextSeq2000, producing 150 bp paired-end reads