Promoter capture-C of EndoC_BH1 cells
Promoter capture-C of EndoC_BH1 cells
Cells were collected and single-cell suspension was made. 10^7 cells were aliquoted in 10ml media. 270ul 37% formaldehyde was added and incubated for 10mins at RT tumbling, then quenched by adding 1.5ml 1M cold glycine (4°C) giving a 12ml total. Sample was tumbled for 5min after glycine addition. After 5min centrifuge at 1000rpm and 4°C the supernatant was removed. The pellet was washed in 10ml cold PBS (4°C), then centrifuged for 5min at 1000rpm and 4°C, then PBS was removed. 35uL of fresh 200x protease inhibitor cocktail were added to 7mL of prepared 3C lysis buffer and mixed thoroughly. The lysis buffer was made by combing 10mM Tris pH8 (500ul 1M), 10 mM NaCl (125ul 4M), 0.2% NP-40 (Igepal) (100ul 100%), and 49.25ml Milli Q in a 50ml Falcon and rotated at 4°C to mix (stored at 4°C). Cell pellet was resuspended in 5ml cold lysis buffer and incubated for 20min on ice, then centrifuged for 5min at 1800rpm and 4°C, then the lysis buffer removed. Pellet was resuspended in 1ml lysis buffer and transferred to 1.5ml Eppendorf prior to snap freezing (ethanol/dry ice or liquid Nitrogen). Cells could be thawed again for digestion at this point or stored at -80°C. The initial 3C libraries were generated following a previously published protocol (Hughes et al., Nature genetics, 2014). The DNA was digested using DpnII, then re-ligated together using T4 DNA ligase, and finally isolated by phenol/chloroform extraction. The 3C libraries were sonicated to an average size of 300bp. Agilent SureSelect library capture protocol was then applied with capture probes targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA transcripts, totaling 36,691 RNA baited fragments. The bait list is provided in the file baits_1frag.bed.