GSM6964773: PCHiC_DNMT_WT_ESC_D0_rep2; Mus musculus; Hi-C
30 million cells were fixed in 2% formaldehyde for 10 minutes and quenched in 0.125M glycine before washing in ice cold phosphate-buffered-saline, scraping and flash freezing. Cells were permeabilized and digested using HindIII (NEB). Restriction sites were filled using Klenow (NEB) in the presence of dCTP, dCGP, dTTP and biotin-14-dATP (Life Technologies). In solution ligation was carried out as described in Nagano et. al, 2015, and DNA was reverse-crosslinked, proteinase K and RNase A treated before purification using phenol/chloroform and ethanol precipitation. Biotin-marked ligation products were isolated with MyOne Streptavidin C1 dynabeads (Life Technologies). Promoter capture (PC) was performed as detailed in Schoenfelder et al., 2015. Briefly, 500 ng of Hi-C DNA was lyophilized using a vacuum concentrator. Pulldown and cleanups were performed as described in the manufacturer's instructions (SureSelect Target Enrichment, Agilent Technologies). The bait system used was as described in Schoenfelder et al., 2015, and included 39,021 biotinylated RNA complementary to the fragment ends of 22,225 mouse gene promoters, consisting of either 1 or 2 RNA baits per promoter overlapping HindIII fragment depending on the complexity of the DNA sequence. Paired-end (PE) adapters were ligated using T4 DNA ligase (NEB) before amplification using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). HiC (promoter capture HiC)