title: Region Capture HiC Imprinted Gene Panel
design: 3-4x107number of 1-7HB2 cells were cross-linked on plate with formaldehyde (Agar Scientific R1026), followed by a quenching step with 1.25M glycine, scraping for cell detachment and two washes with cold PBS 1X. The cell pellet was re-suspended in 50ml freshly prepared ice-cold lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal CA-630 (Sigma-Aldrich, I8896-50ML), one protease inhibitor cocktail tablet (Roche complete, EDTA-free 11873580001). Cells were lysed on ice for a total of 30min, with 2 x 10 strokes of a Dounce homogeniser with a 5-min break between Douncing to minimise cell clumping. Following lysis, the nuclei were pelleted and washed with cold 1.25xNEB Buffer 2 (NEB, B7002S) then re-suspended in 1.25xNEB Buffer 2 to make two aliquots of 1015x106 cells for digestion. Hi-C libraries were digested using 1500U MBOI (NEB, R0147M) at 37C overnight while orbital shaking. Following digestion, the restriction fragment overhangs are filled in for 1h with dNTPs including biotin-14-dATP (Life Technologies, 19524-016). Fragments are then blunt-end ligated with 1U/l T4 DNA ligase (Invitrogen, 15224-025) under dilute conditions to favour ligation between crosslinked fragments (in 15 ml tube for overnight at 16C). DNA crosslinks were then reversed with 10mg/ml proteinase K (Roche, 03115879001) for 6- 8 hours at 65C followed by RNase A (Roche, 10109142001) treatment at 37C for 60 minutes. Two rounds of DNA extraction/purification were carried out with phenol pH 8.0 (Sigma-Aldrich, P4557) and phenol: chloroform: isoamyl alcohol (Sigma-Aldrich, P2069) followed by precipitation with 3M sodium acetate pH 5.2 (Sigma-Aldrich, S7899) and 2.5x volume of ice cold 100% ethanol on wet ice for 1- 2 hours. The Hi-C Librarys quantity and quality were assessed by running 50-100ng of the Hi-C libraries on a 0.8% agarose gel. Hi-C marking and Hi-C ligation efficiency was verified by PCR digest assay using MBOI and CLAI (NEB, R0197S) enzymes. Biotin is then removed from the ends of un-ligated fragments using the exonuclease properties of T4 DNA polymerase (NEB, M0203L) and DNA was sheared to obtain DNA fragments with a peak concentration around 400 bp. DNA ends were repaired and fragments, with internally incorporated biotin, are pulled down using magnetic Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601). After PE adaptors ligation ((5-P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3  and 5-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3), pre-Capture amplication was performed with eight cycles of PCR on multiple parallel reactions from Hi-C libraries immobilized on Streptavidin beads, which were pooled post PCR and SPRI Ampure XP beads (Beckman Coulter, A63881) puried. The nal Hi-C library was re-suspended in 25ul of Tris low-EDTA and quantified by the Qubit dsDNA BR Assay Kit (ThermoFisher, Q32853). The size distribution of the library was assessed by Tapestation D1000 (Agilent). The HiC capture regions were enriched via hybridisation with biotin-RNA probes (Agilent Technologies). The capture regions of interest were then pulled-down with Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601) and purified using SPRI Ampure XP beads (Beckman Coulter, A63881). Finally, Hi-C Capture library was amplified and then sequenced with Illumina 50 bp paired end sequencing.