GSM1464010: ds065_293T_Input; Homo sapiens; ChIP-Seq
IP was carried out as described in Schmidt et al. (2009) (PMID 19275939). In brief, cells were cross-linked using 1% formaldehyde for 10 min at RT, reaction was quenched with 1/20th volume 2.5M Glycine and cell pellets were collected after scraping 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 and 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 was combined with 100ul protein-A magnetic beads preloaded with either 10 ug anti-FOXM1 antibody (GeneTex (USA); cat no. GTX-1000276 and GTX102170) or 10 μl anti-GFP antibody (Abcam, Cambridge, UK; ab290) O/N at 4°C. Beads were washed X6 in RIPA buffer (50mM HEPES pH7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl) and DNA was eluted for 16 hr at 65°C in elution buffer (50mM Tris-HCl, pH8.0, 10mM EDTA, 1% SDS). Extracted DNA was treated with RNase A for 30 min at 37°C, followed by Proteinase K for 1 hr at 55°C, and DNA was purified by phenol-chloroform extraction and quntified by bioanalyzer and quibit. Illumina ChIP-seq kit sample prep kit was used to prepare duplicate/triplicate samples