GSM7158146: MCF10DCIS,P6,cHiC; Homo sapiens; Hi-C
Hi-C was performed as previously described (Belton et al., 2012) and capture probes were designed in collaboration with Agilent. The capture procedure was performed as per the manufacturer's instructions. Cells were fixed with 1% methanol free formaldehyde for 10′ at room temperature, treated with glycine on ice for 15 minutes, PBS rinsed, and snap frozen in liquid nitrogen. HINDIII restriction digested products were blunt ligated after incorporation of biotinylated dCTP. The DNA was sonicated and crosslinks were reversed. Beads were used to pull down biotinylated di-tags. Sheered ends were repaired, A-tailed, and barcoded adapters were ligated. High ligation efficiency was determined by nhel restriction digestion (blunt end formation creates a nheI site). The Belton et al., 2012 protocol was modified to increase the ligation step to 10 hours with a second spike-in of t4 ligase at hour 4. We found a dramatic increase in ligation efficiency using these conditions.