GSM1704493: Region Capture Hi-C, GM12878 Lymphoblastoid cells, replicate one & two; Homo sapiens; OTHER
Cells were thawed on ice and re-suspended in 50ml freshly prepared ice-cold lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal CA-630, one protease inhibitor cocktail tablet). Routinely, two pellets from each cell line were re-suspended and combined in 7ml complete lysis buffer to give ~50-60million cells. Cells were lysed on ice for a total of 30 min, with 2x10 strokes of a Dounce homogeniser with a 5 minute break between Douncing. Following lysis, the nuclei were pelleted and washed with 1.25xNEB Buffer 2 then re-suspended in 1.25xNEB Buffer 2 to make aliquots of 5-6million cells for digestion. Following lysis, Hi-C libraries were digested using HindIII then prepared as described in van Berkum et al with modifications described in Dryden et al. Pre-Capture amplification was performed with 8 cycles of PCR on multiple parallel reactions from Hi-C libraries immobilised on Streptavidin beads which were pooled post-PCR and SPRI bead purified. The final library was re-suspended in 30µl TLE and the quality and quantity assessed by Bioanalyzer and qPCR. Hi-C samples corresponding to 750ng were concentrated in a Speedvac then re-suspended in 3.4μl water. Hybridisation of SureSelect custom promoter and region capture libraries to Hi-C libraries was carried out using Agilent SureSelectXT reagents and protocols. Post-capture amplification was carried out using 6 cycles of PCR from streptavidin beads in multiple parallel reactions, then pooled and purified using SPRI beads.
GEO Accession
GSM1704493