MCF7 cells were purchased from ATCC and grown in estrogen-depleted media for 3 days and treated with 10 nM of 17β-estradiol for 45min as performed in (Shang and Brown, 2002). A 3C library was then prepared as previously described (Pomerantz et al., 2009). Briefly, after hormone treatment, MCF7 cells were trypsinized, fixed with 1% formaldehyde and, lysed with 3C lysis buffer (10 mM TrisHCl pH 8, 10 mM NaCl, 0.2% Nonidet P-40). The pelleted cell nuclei were resuspended in restriction butter with SDS (final concentration 0.1%) and incubated for 10 min at 65°C. After then, TritonX-100 (final concentration 1%) and HindIII 160 U per 1 × 106 cells were added and incubated for 24 hr at 37°C on a rotating platform. The digested samples were added to the 3C ligation mix buffer with T4 DNA ligase and incubated for 24 hr at 16 °C. The ligated samples were decrosslinked at 65 °C with proteinase K for overnight and were Phenol/Chloroform extracted, EtOH precipitated and desalted. Target primers were designed against HindIII digested fragments within or close to DHS around SNPs
