ChIP-Atlas: SRX1133071

Antigen

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: RNA-Seq: Polyadenylated RNA was isolated using Olig dT beads (Invitrogen). RNA was fragmented to a size of 200-600 base pairs, followed by ligation to RNA adapters using T4 RNA Ligase (NEB). Adapter primers were used for cDNA synthesis, followed by RNA degradation. Each cDNA library was ligated to a DNA adapter, used for barcoding and PCR enrichment of the library. Libraries were pooled and sequenced on the Illumina HiSeq 2000. ChIP-Seq: Cells were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads (Invitrogen) for 2-3 hour at 4°C, with constant agitation Immunoprecipitated DNA was end repaired using the End-It DNA End-Repair Kit (Epicentre), extended using a Klenow fragment (3’-5’ exo) (NEB), and ligated to sequencing adapter oligos (Illumina). Each library was then PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300-600 was selected for sequencing on the Illumina HiSeq 2000.