GSM2067354: MCF7_N2; Homo sapiens; OTHER
Capture-C libraries were prepared as previously described (Davies et al. 2016 Nature Methods). Cells were fixed with 2% (vol/vol) formaldehyde for 10 min, quenched with 125 mM glycine in PBS and then lysed in cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, 0.2% Igepal, 1× cOmplete Protease Inhibitor Cocktail (Roche). Chromatin was digested with Dpn2 (New England Biolabs) at 37oC overnight.  Fragments were then diluted and ligated with T4 DNA ligase (Thermo Scientific) at 16oC overnight. Crosslinking was reversed by overnight incubation at 60oC with proteinase K (Bioline). The 3C libraries were then purified by phenol-chloroform and chloroform extraction followed by precipitation in ethanol at -80oC overnight. Digestion efficiency was determined by qPCR and gel electrophoresis. Sequencing libraries were prepared from 5 μg of each 3C library by sonication using a S220 focused-ultrasonicator (Covaris) to a average size of 200bp and indexed using NEBnext reagents (New England Bioloabs) according to protocol. Enrichment of 1-2 μg of indexed library incubated with 13 pmol of a pool of biotinylated oligonucleotides (Intergrated DNA technologies or Sigma) was performed using the SeqCap EZ system (#06953212001, Roche/Nimblegen) following the manufacturer’s instructions. Two rounds of capture employing 48-72 hr and 24 hr hybridizations respectively were used. Capture enrichment was determined by qPCR. Correct library size was confirmed using a Bioanalyzer DNA 1000 or Tapestation D1000 kit (Agilent), and DNA concentrations were determined using a Qubit 2.0 Fluorometer (ThermoFisher Scientific).
GEO Accession
GSM2067354