GSM8458825: K562, WT; Homo sapiens; Hi-C
Hi-C template preparation was performed as previously described (Rao et al. Cell 2014) with modifications as follows: ten million cells per sample were harvested and fixed in 2% formaldehyde; fixation was quenched by glycine at final concentration of 0.2 M. Four-cutter restriction enzyme MboI (NEB, cat. no. R0147) was used for in situ digestion (400 U/10 million cells). Digested DNA ends were labeled with biotin-14–dATP (Invitrogen, cat. no. 19524016) in a Klenow end-filling reaction. End-repaired, ligated, and reverse-crosslinked DNA was purified using isopropanol and magnetic beads (Macherey-Nagel NucleoMag PCR Beads). Purified DNA was sheared to 300-500 bp with Covaris, following manufacturer's instructions. After AMPure XP size selection, appropriately-sized ligation fragments marked by biotin were immobilized using Dynabeads MyOne Streptavidin C1 (Invitrogen, cat. no. 65001) and ligated to indexed KAPA UDI Adapters (Roche). The non-amplified, immobilized and indexed Hi-C library was subsequently used as input for target-specific capture using RNA baits. Capture Hi-C of the targeted genomic sequences was carried out with KAPA HyperCap (Roche) with the bait library according to manufacturer's instructions (Roche). The total output of post-capture was amplified with 12 cycles We designed a custom biotinylated KAPA HyperCap Target Enrichment Probes (Roche) library. Biotinylated 120-mer RNA baits were designed to target MboI restriction fragments within the TPCN2-LTO1 region (hg38: chr11: 68,998,932-69,725,322) and a control region (chr18:21,427,907-22,925,680) For each MboI motif two probes were designed, each covering the MboI recognition motif and extending away from the motif in opposite directions. Probes were filtered using Roche Hyperdesign standard selection parameters (15 mer frequency of 1000, maximum allowed close matches to hg38 of 20). Capture Hi-C