title: GSM1143123: Med12_MCF7; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP and FAIRE assays, DNA was extracted according to Svotelis et al. (Methods Mol Biol., 2009) and to Eeckhoute et al. (Genome Res., 2009) with few modifications. In brief, ~20-30.106 cells were cross-linked using 1.1% formaldehyde for 10 or 30 min at RT in 20ml of PBS 1X, reaction was quenched with 1ml of 2.5M Glycine. Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCI of pH 8.1) during 30 min at 4°C and sonicated to have DNA fragment size between 200 and 300 bp. For ChIP, lysate was incubated overnight at 4°C on a rotator with 5µg of antibody. 35 µl of protein-A magnetic beads (Invitrogen) were added during 2h before washing with TSE-150 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris–HCl of pH 8.1, 150 mM NaCl), TSE-500 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris of pH 8.1, 500 mM NaCl), LiCl detergent (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris of pH 8.1) and three times with TE 1X. For FAIRE, soluble chromatin was subjected to three consecutive phenol-chloroform extractions. DNA was then reverse-crosslinked overnight at 65ºC and treated with RNAse A for 30 min at 37°C, followed by Proteinase K for 2 h at 37°C. After purification, DNA was quantified with Picogreen (Invitrogen) before library preparation. Libraries were prepared according to Illumina's instructions. Briefly, 10 ng (ChIP-seq) or 50 ng (FAIRE-seq) of DNA were end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase (Enzymatics). DNA fractions of ~300 bp were selectively isolated with solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure, Beckman Coulter). After adapter ligation, DNA was PCR amplified with Illumina primers, the quality and quantity of each DNA library was analyzed using Agilent Bioanalyser before sequencing on HiSeq (Illumina) following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1143123