GSM1575699: OIS input replicate 1; Homo sapiens; ChIP-Seq
Cells were cross-linked with 1% formaldehyde in PBS at room temperature for 10 min, then quenched in 125 mM glycine in PBS at room temperature for 5 min.  The cells were washed by cold PBS once and were collected by centrifugation and sonication in lysis buffer (50mM Tris pH 7.9, 10 mM EDTA, 1% SDS, 1x protease inhibitor cocktail and 1mM DTT) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation 14,000rpm for 10min at 4 °C, and 20 µl supernatant was used as input for quantitation, the remaining supernatant was diluted 10-fold in dilution buffer (20 mM Tris pH 7.9, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 1X protease inhibitor cocktail and 1mM DTT), and pre-cleared by incubation with protein A-agarose beads at 4°C for 3hr. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions at 4°C overnight with an antibody against macroH2A1, then incubated with protein A-agarose beads at 4°C for 2hr. No antibody negative controls were included for every ChIP experiment. The immuneprecipitated DNA was cleared of protein by digestion with 0.4mg/ml glycogen and proteinase K (2.5U/ml, Roche) in Txn stop buffer (20 mM EDTA, 0.2 M Nacl and 1% SDS) at 37°C for 1 hour. The DNA was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated. DNA fragments from ChIP reactions were repaired using T4 DNA Polymerase (NEB) and T4 DNA Polynucleotide kinase (NEB) in T4 DNA ligase buffer (NEB). Next, dA ends were generated by employing Klenow Fragment (3’->5’ exo-, NEB), in Buffer 2 (NEB). Adapter ligation reactions were then set up using indexed adapters and Quick Ligase (NEB). Ligation-mediated PCR was then performed using Phusion High-Fidelity DNA Polymerase (NEB). The libraries were visualized on 2% E-Gel® General Purpose Agarose Gels (Invitrogen). Sequencing for the macroH2A1 ChIPs were performed on an Illumina HiSeq 2500 at the Einstein Epigenomics Core Facility. Sequences reads were mapped to the human genome version hg19 using ELAND version 1.7.0 (Illumina).
GEO
GSM1575699