GSM6031048: H9,rep1; Homo sapiens; Hi-C
In situ Capture HiC was performed as previously published (Davies 2016) with the following modifications. Cells were crosslinked with 2% formaldehyde. In short, 10x10⁶ cells were lysed on ice in 10 mM Tris-HCL pH8.0, 10 mM NaCl, 0,2% NP40, 1X complete protease inhibitor cocktail (Roche). De-crosslinking was performed in 100 µL 0,5% SDS at 37°C for 1h under agitation (1400 rpm). Nuclei permeabilization was done by adding 50 µL of 10% Triton X-100 and 290µL H2O and incubated for 15 minutes at 37°C and swirling at 1400 rpm. To prepare for digestion, 50µL of 10X DpnII buffer was added and 50µL were taken as an undigested control. At mid day, a first 10µL of DpnII (NEB, 50 000 U/ml) was added and incubated for 4 hours at 37°C, swirling at 1400 rpm. Another 10 µL was added and incubation was carried on overnight. A final 10µL of DpnII  was added the following morning and  left for 4 hours. DpnII was inactivated by heating at 65°C for 20 minutes and 50 µL were taken as an digested-unligated control. Ligation was carried by adding 800 µL of ligation cocktail containing 240U T4 ligase (ThermoScientific 30U/µL), 1X T4 ligase buffer, and incubated overnight at 16°C swirling at 1000 rpm. To prepare for DNA purification, samples and controls were incubated with 150µg of ProteinaseK (Eurobio) for 4 hours ar 65°C. Then, 0,1 vol. of NaAc 3M and 3 vol. of 100% EtOH were added and incubated overnight at -80°C. Precipitated DNA were pelleted by centrifugation, washed with 70% EtOH and finally air dryied. Samples and controls were resuspended in 100 µL and 20 µL H2O respectively and 1 µL of RNAse A (ThermoScientific) was added and incubated at 37°C for 1 hour.  DNA quantities in sample and controls were determined using the Qbit broad range kit (ThermoScientific) and 100 ng of each were loaded on an agarose gel as quality control of the digestion and ligation steps.