title: GSM2259751: Day3 Smad3CA+ Dox+ Oct4 Rep3; Mus musculus; ChIP-Seq
library_construction_protocol: A pellet of ~2x10^6 cells was thawed and nuclei were isolated by re-suspension in lysis buffer (5 mM Pipes pH 8.0, 85 mM KCl, 1% NP40) with fresh protease inhibitors (complete protease inhibitor cocktail, Roche) for 20 minutes on ice followed by brief vortexing and centrifugation at 500 g for 10 minutes at 4 °C. The nuclear pellet was re-suspended in 300 ul IP buffer (0.5% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, fresh protease inhibitors) and sonicated to produce ~200-300 bp fragments in a BioRuptor Sonicator with a total of 5-8 cycles of: 10 pulses of sonication on the “high” setting, each followed by 30 seconds off. Thus, each sample received between 50-80 pulses of 30-second 'high' sonication. Samples were then diluted in IP buffer without SDS (to final 0.1% SDS), then 8 µg antibody was added for overnight incubation, rotating at 4 °C. ChIP'd DNA from ~2x10^6 cells was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters, which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for total 16-18 cycles and library fragments of ~250 bp were isolated with SeraMag beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2000 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM2259751