title: GSM3399737: ACTA2R179H_IPH3K27ME3; Homo sapiens; ChIP-Seq
library_construction_protocol: Human wild-type or aneurysm (ACTA2R179H)10 smooth muscle cells (10 million) were fixed with 1% of formaldehyde at 37 C for 20 min, quenched with 125 mM glycine for 5 min (RT) and protein lysates were prepared using EpiTect ChIP kit according to the manufacturer's instructions (Qiagen, USA). Next, total protein lysates were sonicated to shear chromatin to an average length of 500–1,000 bp, followed by centrifugation for 10 minutes at max speed. Supernatants were collected into a 2 mL tube containing 6 μg of monoclonal antibody against HDAC9 (ab59718), BGR1 (ab110641), H3K27me3 (Abcam, ab4729) or IgG isotype control (ab171870) and 1 ml of lysis buffer supplemented with 1× of protease inhibitor cocktail (Roche Diagnostics), followed by incubation overnight (14 hours) at 4°C. Immunoprecipitates were analyzed using EpiTect ChIP kit according to the manufacturer's instructions (Qiagen). Then, ChIP-ed DNA was quantified and used for DNA-end repair (3'-dA) followed by PCR amplification and size selection (usually 100-400bp, including adaptor sequence). The, qualified libraries were used for HiSeq sequencing (Illumina HiSeq 50 SE sequencing).
attributes.tag: GEO Accession
attributes.value: GSM3399737