mouse embryonic stem cells cell-line 2: Hi-C
Mouse embryonic mutant (ES-TKO; DNMT1-/-, DNMT3a-/-, DNMT3b-/-) and wildtype stem cells (ES-WT) were cultured without feeders on 0.2% gelatine-coated dishes in DMEM with non-essential amino acids, supplemented with 15% fetal calf serum, 2 mM L-glutamine, LIF and 0.001% ß-mercaptoethanol (37 °C, 7% CO2)15. 106 embryonic stem cells were centrifuged (300 x g for 5 minutes), then resuspended in 1xPBS (1x106 cells per mL). Fresh formaldehyde was added to a final concentration of 1% and incubated for 10 min at RT. The crosslinking reaction was quenched by adding glycine (0.25 M final concentration). After subsequent washing, the pellets were centrifuged for 5 minutes at 300 x g at 4°C and flash-frozen in liquid nitrogen. Cells were processed directly or stored at -80°C. Cells were lysed with 300 µL cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, freshly added protease inhibitor) for 15 min on ice and then centrifuged at 2500 x g for 5 minutes. Pelleted nuclei were washed with 500 µL lysis buffer, then permeabilized and resuspended in 50 µL of 0.5% SDS. Nuclei permeabilization was carried out for 10 min at 62°C under constant rotation. After adding of 145 µL water and 25 µL 10% Triton X100, the nuclei were incubated at 37°C for 15 minutes. Then, 25 µL of Restriction Enzyme buffer (NEB DpnII-buffer) and 400 U of restriction enzyme (DpnII, NEB) were added. Restriction was carried out at 37°C over night under constant rotation. DpnII restricted samples were heat inactivated at 62°C for 10 minutes. Marking of DNA-ends, proximity ligation and decrosslinking were performed as described in Rao et al. (Cell, 2014). Two DNA precipitation steps were performed after decrosslinking including RNase A digestion (37°C, 30 min) after first precipitation. The ligated DNA was resuspended in 100 µL water and sheared using 30 cylces of Bioruptur (Diagenode, 30 s 'on', 90 s off, 'low'). Size selection using Ampure Beads XP (Beckman), Biotin pull-down and library preparation were carried out as described by Rao et al. (Cell, 2014). To reduce duplicates, a 1:4 dilution of T1-beads (Invitrogen) containing the Hi-C library was taken to determine minimum number of cycles for final library amplification (KAPA SYBR FAST qPCR, Kapabiosystems). The final amplification was performed in technical replicates using the rest of the undiluted T1 beads using Phusion High-Fidelity DNA polymerase (NEB). PCR products of technical replicates were pooled and cleaned twice using Ampure Beads XP (Beckman, 0.9 x beads).