title: GSM3533471: ChIP-Seq_MCF7_Med12_siCTL_E2; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP assays, cells were maintained in stripping medium (phenol red free) for three days before treating with or without estrogen (E2, 10-7 M) for 1 hr. Cells were then fixed with 1% formaldehyde (Sigma) for 10 mins at room temperature (RT) (for MED12 ChIP), or fixed with disuccinimidyl glutarate (DSG) (2 mM) (Proteochem) for 45 mins at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 10 mins at RT (for CARM1 ChIP). Fixation was stopped by adding glycine (0.125 M) and incubated for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300~500 bp average in size through sonication. Resultant was immunoprecipitated with anti-MED12, anti-Pol II or anti-CARM1 antibody overnight at 4 ℃, followed by incubation with protein G magnetic beads (Bio-Rad, 161-4023) for an additional 2 hrs. After washing and elution, the protein-DNA complex was reversed by heating at 65 ℃ overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to high throughput sequencing. The libraries were constructed following NEB's Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.
attributes.tag: GEO Accession
attributes.value: GSM3533471