alias: GSM3530163
title: GSM3530163: E14-2i_1; Mus musculus; Hi-C
library_construction_protocol: Hi-C was performed largely as described (Rao et. el., 2014) with minor modifications. Briefly, ES cells were crosslinked in 1% formaldehyde for 10 minutes, snap-frozen in 2-5 mln aliquots and stored at -80°C. After permeabilization in lysis buffer (0.2% Igepal, 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1x Halt Protease inhibitor cocktail) nuclei were isolated in 0.3% SDS in NEBuffer 3 at 62° for 10 minutes. SDS was quenched with 1% Triton X-100 at 37° for 1 h, then the nuclei were pelleted and resuspended in 250 ul DpnII buffer with 600 U DpnII. After overnight digestion, 200 more units were added for 2 hours. Then the ends were filled-in using Klenow, d(G/C/T)TPs and biotin-14-dATP for 1.5 hrs at 37°. After ligation at room temperature for 4 hrs the nuclei were spun down, resuspended in 200 ul mQ and digested with proteinase K for 30 min at 55° in presence of 1% SDS. Then the cross-links were reversed at 65° overnight after addition of NaCl to a final concentration of 1.85 M. In the morning, after ethanol precipitation and a wash with 70-80% Ethanol, DNA was resuspended in 500 ul of sonication buffer (50 mM Tris pH 8.0, 0.1%SDS, 10 mM EDTA), incubated on ice for 15 min and then sheared using a probe sonicator to fragment size of 200-700 bp. Then the DNA was concentrated on Amicon filter units, bound to MyOne T1 Streptavidin beads and used for Illumina library preparation. Small aliquots were taken before and after DpnII treatment, and before sonication to confirm efficient DNA digestion and ligation by running them on 1% agarose gel. Samples were first test-sequenced on NextSeq 550 (WTCRF, Edinburgh) to check library quality, and then selected libraries were sequenced deeply on HiSeq 4000 (BGI). In house Illumina library protocol from Rao et. al., 2014.
identifiers.namespace: GEO
identifiers.id: GSM3530163