GSM7665191: Human fetal neural stem cell (hfNSC) cell line, ChIP for H3K27ac, replicate 1; Homo sapiens; ChIP-Seq
hiPSC, hiPSC-NSC and hfNSC were cross-linked for 10 min at RT with 1% methanol-free formaldehyde-containing medium and blocked with 0.125 M glycine. Cross-linked cells were washed with PBS containing protease and phosphatase inhibitors and stored at -80 until use. Nuclei were collected upon cell lysis using Cell lysis buffer and by nuclei release with B tight dounce. Nuclear extracts were sonicated on Diagenode 1.5 ml tubes with BioruptorPico in sonication buffer for 12 (for hiPSCs) or 15 (for hiPSC-NSCs and hfNSCs) cycles of 30 sec ON /30 sec OFF to obtain DNA fragments averaging 200 bp in length and then checked by microcapillary electrophoresis on 2200 TapeStation instrument (Agilent Technologies). Chromatin preclearing was performed using IgG isotype (1 ug IgG/106 cells)-Protein G Agarose coated beads (Thermo Scientific) mixed to chromatin. The equivalent of 3-6 x107 cells were precipitated overnight with 1 ug IgG/106 cells of rabbit antibody against H3K27ac (ab4729, Abcam). Before antibody precipitation, total chromatin was collected as INPUT. H3K27Ac chromatin was precipitated with precleared (RIPA Buffer + protease inhibitors; BSA at 0.5 ug/ul and S256 containing salmon sperm at 2.7 ug/ul overnight at 4°C)-Protein G Agarose coated beads for 2 hours on rotating wheel at 4°C. Beads were then washed with RIPA buffer, LiCl buffer and TE buffer. Chromatin was then eluted with Elution Buffer, reverse cross-linking was performed with 0.3M NaCl and Rnase A (10 mg/ml) for 4-5 hrs at 67°C. Chromatin was precipitated overnight on 100% ethanol with glycogen (5 mg/ml) at -20 °C. After protein digestion (PK Buffer and Proteinase K (20 mg/ml) 2 hrs at 45°C), DNA was purified with Qiaquick PCR purification kit (Qiagen, Germany). ChIP-seq libraries were prepared from 10 ng of immunoprecipitated DNA (ChIP) and control DNA (INPUT: nuclear extracts sonicated but non-immunoprecipitated) using the ChIP-seq DNA sample preparation kit (Illumina). Libraries were checked by microcapillary electrophoresis on Bioanalyzer 2100 (Agilent Technologies), using with the High Sensitivity DNA Assay kit, and quantified with Quant-iT™ PicoGreen dsDNA Kit (Invitrogen) on Nanodrop Fluorometer. Each library was sequenced in one lane of a single-end 50-cycle Illumina run.