FAIRE-seq
FAIRE-seq
U2OS cells, were grown in DMEM supplemented with 10% foetal bovine serum. FAIRE was performed as described previously (Giresi et al., 2007; Eeckhoute et al., 2009) with slight modifications. 5 x 107 U2OS cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The same amount of non-cross-linked cells was used as control. Glycine was added to a final concentration of 125 mM and the cells were rinsed with cold PBS and harvested by scraping into cold PBS containing protease inhibitors. Cells were then lysed with lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl at pH 8.0, 1 mM EDTA) containing a protease inhibitor cocktail (Roche) and sonicated for 15 min (30 sec on/off cycles) using a Bioruptor (Diagenode) set up at the highest intensity. After centrifugation at 13,000g for 10 min at 4C to precipitate cellular debris, 50 ul lysate were taken as input control. The soluble chromatin was isolated and subjected to three consecutive phenol-chloroform extractions (Sigma, P3803). Each time, the aqueous phase was recovered and mixed with an equal volume of phenol-chloroform. After vortexing, the mixture was centrifuged at 13,000g for 5 min and the aqueous phase was recovered. Samples were then incubated overnight at 65C to reverse cross linking. DNA was finally purified using a PCR purification kit (QIAGEN). DNA were quantified using a Nanodrop and 5 ng DNA was used as qPCR templates.
Experimental Factor: INDIVIDUAL
faire