GSM1528694: jc379_MCF_input_rep3_CRI01; Homo sapiens; ChIP-Seq
6 x15cm2 plates used per IP protocol, IP carried out as described in Schmidt et al (2009). In brief, cells were cross-linked using 1% formaldehyde for 10 min at RT, reaction quenched with 1/20th volume 2.5M Glycine and cell pellets collected after scrapping plates. Nuclear lysates were prepared by lysing with LB1 (50mM Hepes-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton-X plus PI) 10 min at 4°C, LB2 (10mM Tris-HCl, pH8.0, 200mM NaCl, 1mM EDTA, 0.5M EGTA plus PI) 5 min at 4°C followed by sonication in LB3 (10mM Tris-HCl, pH8.0, 100mM NaCl, 1mM EDTA, 0.5M EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine plus PI) using a diagnode sonicator to give a fragment size of ~200-300bp. Sonicated lysate combined with 100ul protein-A magnetic beads preloaded with 10ug anti-FOXM1 antibody (Santa Cruz Biotechnology, USA; cat no. sc-502; lot no. D0110) O/N at 4°C. Beads washed X6 in RIPA buffer (50mM HEPES pH7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl) and DNA eluted for 16 hr at 65°C in elution buffer (50mM Tris-HCl, pH8.0, 10mM EDTA, 1%SDS). Extracted DNA treated with RNase A for 30 min at 37°C followed by Proteinase K for 1 hr at 55°C and DNA purified by phenol-chloroform extraction. DNA prepared for Illumina library construction by repair with Klenow and T4 polymerase, A-tailing with Klenow exo polymerase and attachment of adapters (diluted 1:20) using DNA ligase. Adapter ligated fragments amplified by PCR using phusion polymerase (12 cycles) and ~200-300 bp fragments extracted by gel purification. Library size and quantity determined by bioanalyser.
GEO Accession
GSM1528694