title: GSM7884007: hNSC, CHD8, ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: hNSCs were double crosslinked by incubation with 2 mM disuccinimidyl glutarate solution, followed by an incubation with 1% of buffered formaldehyde solution. Subsequently, the cell pellets were lysed with lysis buffer (1% SDS, 50 mM TrisHCl pH 8.1, 10mM EDTA pH 8.0 and 1x CEF protease inhibitors (Roche)) and sonicated using a Bioruptor Pico sonication device (Diagenode, B01060001) for 14 cycles of 30sec on and 30sec off. The ChIP was performed on the resulting sheared chromatin by incubation overnight at 4°C with ProtG Dynabeads (Invitrogen, #100-04D) that had been pre-coupled to either the CHD8 antibody, TRRAP antibody or IgG antibody (Diagenode, C15410206) as control. Following the overnight incubation, the Dynabeads were washed twice with Low salt wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl), once with High salt wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 500 mM NaCl), once with LiCl wash buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 0.5% IGEPAL, 1% NaDeoxycholate) and once with T10E1 (10 mM TrisHCl pH 8.0, 1 mM EDTA). The bound complexes were eluted of the beads by incubation for 1 hour at 65°C in ChIP elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) with occasional gentle vortexing. The samples were de-crosslinked overnight by incubation at 55°C. DNA was extracted from the elutions by the Phenol:Chloroform:Isoamyl alcohol (PCI) method and diluted in water. The DNA libraries for the input, IgG, CHD8 and TRRAP ChIP samples were prepared using the ThruPLEX V2 DNA sample preparation protocol from Takara Bio.