GSM2872710: Normoxia_DNase-seq; Homo sapiens; DNase-Hypersensitivity
DNase-seq was performed as described previously (Winter et al., 2013). Briefly, SK-N-BE(2)c cells were resuspended in RSB buffer (10 mM Tris pH 7.4, 10 mM NaCl, and 3 mM MgCl2) and lysed by slowly adding lysis buffer (0.1% IGEPAL CA-630 in cold RSB buffer). The nuclei were collected by centrifugation at 500 g for 5 min and were then digested with optimal concentration of DNase I (Roche) at 37°C. After 15 min, the digestion was stopped by adding EDTA. To remove the RNA, the samples were treated with RNase A at 37°C for 30 min, followed by proteinase K treatment at 55°C overnight. Subsequently, the DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNase I ends were blunted and ligated to biotinylated linkers containing an MmeI restriction site. After digestion with MmeI, the DNase I digested ends were enriched using streptavidin magnetic beads (Dynabeads M-280, Life Technologies) and ligated to a second set of linkers. Subsequently, the DNA fragments were lightly amplified by PCR and sequenced using HiSeq technology (Illumina).