GSM2501569: LM2_EZH2_ChIPSeq; Homo sapiens; ChIP-Seq
Cells were harvested by crosslinking with 1% formaldehyde in cell culture medium for 15 minutes at room temperature. After quenching with the addition of 125 mM glycine for 5 minutes at room temperature, the cells were washed twice with ice cold PBS. After aspiration of all liquid, pellets consisting of ~10E7 cells were flash frozen and stored at -80C. Fixed cells were thawed and sonicated to obtain chromatin fragments of ~200 to 700 bp with a Branson 250 Sonifier. Solubilized chromatin was immunoprecipitated with ~5ug antibody against c-Myc (Santa Cruz; sc-764), Fra-1 (Santa Cruz; sc-183), E2F1 (Millipore; 05-379) and p53 (BD-Pharmingen; 554294). Immunoprecipitation was performed retaining a fraction of input 'whole-cell extract' as a control. Antibody-chromatin complexes were pulled-down using Dynabeads Protein G, washed and then eluted. After crosslink reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol, precipitated in ethanol and treated with RNase. ChIP DNA was quantified by fluorometry using the Qubit assay (Invitrogen). For each ChIP or control sample, ~5ng of DNA was used to generate a standard Illumina sequencing library. Briefly, DNA fragments were end-repaired using the End-It DNA End-Repair Kit (Epicentre), extended with a 3'  'A' base using Klenow (3' 5' exo-, 0.3 U/ul, NEB), ligated to standard Illumina adapters (75 bp with a 'T' overhang) using DNA ligase (0.05 U/ul, NEB), gel-purified on 2% agarose, retaining products between 275 and 700 bp, and subjected to 18 PCR cycles. These libraries were quantified by fluorometry and evaluated by quantitative PCR to confirm representation and specific enrichment of DNA species. Libraries were sequenced in one or two lanes on the HiSeq 2000 using standard procedures for cluster amplification and sequencing by synthesis.