title: GSM3521317: ChIP-seq_WT_Cas9; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assays were performed as described previously (Rada-Iglesias et al., 2011). Briefly, approximately 10&7 NCCIT cells were fixed in 1% formaldehyde for 10 minutes at room temperature in PBS, then quenched with glycine to a final concentration of 0.125 M for 10 minutes. Chromatin was sonicated to 0.5-2.0 kb using Bioruptor (Diagenode, Liège, Belgium), cleared by centrifugation, divided into separate aliquots for each antibody, and incubated with 5 mg of antibody overnight at 4° C. Subsequently, 100 mL of Dynabeads protein G (Thermo Fisher Scientific) were added to the ChIP reactions and incubated for 4-6 hours at 4° C. Magnetic beads were washed and chromatin was eluted, followed by reversal of crosslinks overnight at 65° C, proteinase K and RNase A treatment, and DNA purification by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. ChIP DNA was resuspended in water. Libraries were prepared using Ovation Ultralow System V2 UDI (NuGEN Technologies, San Carlos, CA, USA) according to manufacturer's instructions, starting with 10 ng of ChIP DNA. Library DNA was analyzed on Bioanalyzer DNA HS (Agilent, Santa Clara, CA, USA), then pooled and sequenced on a HiSeq 4000 (Illumina, San Diego, CA, USA) at the Stanford Genome Sequencing Service Center, using 2x150 sequencing with dual index read.
attributes.tag: GEO Accession
attributes.value: GSM3521317