GSM1552414: rsH1-H3K27me3; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde at 37C for 10min, quenched with glycine at 37C for 5 min. Fixed cells were sonicated using Epishear (Active Motif) to achieve 200-700 size chromatin fragments. Solubilized chromatin was immunoprecipiated with antibody against H3K4me3 (Abcam 8580) and H3K27me3 (Millipore 07-449). Antibody-chromatin complexes were pulled down using Dynabeads protein A (Invitrogen), washed and then eluted. After cross-linking reversal, RNase and proteinase K treatment, immunoprecipiated DNA was purified using AMPure beads (Beckman Coulter). ChIP DNA were end-repaired and 5' phosphorylated using T4 DNA Polymerase, Klenow and T4 Polynucleotide Kinase (Enzymatics). A single Adenine was added to 3' ends by Klenow (3-->5' exo-), and double-stranded Bioo Illumina Adapters (Bioo Scientific) were ligated to the ends of the ChIP fragments. Adapter-ligated ChIP DNA fragments were subjected to 15 cycles of PCR amplification using Q5 polymerase (NEB). AMPure beads were used to purify DNA after each step (Beckman Coulter). Pooled libraries were sequenced on the NextSeq 500 for single-end 75bp using high-output flowcell or on the HiSeq 2000 for single-end 36bp according to manufacturer's instructions.
GEO Accession
GSM1552414