GSM5608848: FL_HiC_rep1; Mus musculus; Hi-C
E14.5 Meckel's cartilage bars were dissected from wild-type C57BL/6J  mouse embryos as previsouly described (Ishizeki, 1996 & Wiszniak, 2015) with modifications. E14.5 forelimb was dissected from wild-type mouse embryos with removal of surrounding connective tissues. Dissected forelimb cartilage was then digested following the same procedure as the digestion of Meckel's cartilage. Briefly, cells were crosslinked in 1% formaldehyde for 10 mins at room temperature and quenched with 0.2M glycine. Cells were pelleted, washed with 1ml HBSS and then resuspended with 1ml ice-cold Hi-C lysis buffer, incubated on ice for 30min and were further dounced with pestles to insure complete lysis. Chromatin was then opened up at the presence of 0.5% SDS at 65°C for 5 mins and quenched with Triton X-100 at the final concentration of 1%. Samples were then digested overnight with DpnII at 37°C on a thermomixer with interval shaking, followed by heat inactivation of DpnII and fill-in of the restriction fragment overhangs. Samples were then ligated with T4 DNA ligase at 16°C for 4hr, and crosslink reversal was performed by Proteinase K digestion. 3C libraries were then extracted and purified by phenol-chloroform isoamyalcohol extraction and ethonal precipitation. Efficiency of digestion and ligation was assessed by gel electrophoresis of quality controls. 3C libraries were then sheared to 200-700bp by Covaris sonication and Dynabeads MyOne Streptavidin T1 was used to perform biotin pull-down after washing. End repair, dA-tailing and ligation of libraries were performed with NEBNext Ultra II Ligation Module. Libraries was amplified on the streptavidin beads using Phusion DNA Polymerase, size-selected with AMPure XP beads, quantified by Qubit dsDNA High Sensitivity Assay and then sequenced on the NovaSeq platform.