mouse fetal cardiac myocyte nuclei rep1: Hi-C
The in situ Hi-C method was adopted from Rao et al. (Cell, 2014). Up to 230,000 FACS-sorted cardiomyocyte nuclei were centrifuged (1000 x g, 5 min, 4°C), washed with cold PBS and resuspended in PBS. Nuclei then were fixated using PFA (1 % final concentration) for 10 min at RT under constant rotation. Fixation was stopped with 2.5 M glycine (final concentration: 0.25 M). Nuclei were centrifuged, washed with PBS (1000 x g, 5 min, 4°C) 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 buffer 2.1 respectively DpnII-buffer) and 400 U of restriction enzyme (HindIII respectively DpnII, NEB) were added. Restriction was carried out at 37°C over night under constant rotation. Both, HindIII and 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).