GSM4314848: H9_ESC_CaptureC_EC1.35enhancer; Homo sapiens; OTHER
Capture-C was performed as previously described (Davies et al., 2015). Briefly, cells were crosslinked for 10 min in 2% formaldehyde in PBS, quenched with 125mM glycine for 5 min, scraped, collected and pelleted by centrifugation (500 rcf, 5 min, 4°C). Cells were washed with 5mL cold PBS, pelleted and resuspended in 5mL cold lysis buffer (10mM Tris pH8, 10mM NaCl, 0.2% Ipegal CA-630 in water with cOmplete Protease Inhibitor Cocktail) for 20 min. Cells were pelleted, washed with 5mL cold PBS, pelleted, resuspended in 1mL cold PBS, flash frozen in liquid nitrogen and stored at -80°C. For 3C library preparation, samples were defrosted on ice, pelleted and resuspended in 1xDpnII digestion buffer before extended digestion with DpnII overnight with addition of extra enzyme. DpnII was then heat inactivated at 65°C and samples were ligated with T4 Ligase for ~22 hours. DNA was extracted by sequential Proteinase K and RNase digestion followed by phenol-chloroform isoamylalcohol extraction and precipitation with ethanol and sodium acetate. Efficiency of digestion and ligation was assessed by gel electrophoresis and digestion efficiency was further assessed by qPCR. For addition of Illumina sequencing adaptors, samples were sheared by Covaris sonication and purified by XP SPRI bead clean-up. Sequencing adaptors were annealed using NEB Ultra II kit. Libraries were PCR amplified using Herculase II polymerase (Agilent) in duplicate to add indexing primers and purified by XP SPRI bead clean-up. Samples were pooled and quantified using Qubit dsDNA BR assay kit.    Biotinylated oligos for capture were designed using the Capsequm online tool (http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi) and ordered from IDT. 1-2μg indexed 3C library was mixed with 5μg COT DNA, 1nmol TS-HE Universal Oligo and 1nmol of TS-HE Index Oligo (Nimblegen SeqCap EZ HE-oligo and Accessory kit) and dried by vacuum centrifugation at 55°C until completely dry. The 3C library plus blocking oligos were then carefully resuspended in 7.5μL 2X hybridization buffer and 3μL Hybridization buffer A and denatured at 95°C for 10min. The 3C library was then transferred into a preheated PCR tube at 47°C containing 4.5μL of pooled Biotinylated oligonucleotide capture probes at 2.9μM. After a brief mix and centrifugation, the 3C library oligo mix was incubated at 47°C for 18-20 hrs. Following this incubation, the 3C library was washed and recovered by streptavidin bead (M-270 Dynabeads, Invitrogen) pull down using the Nimblegen SeqCap EZ Hybridization and wash kit. Following the recovery of the captured material, the captured DNA was amplified on the streptavidin beads using KAPA HiFi HotStart ReadyMix and POST-LM PCR oligo 1&2 and purified by XP SPRI bead clean-up. For improved capture efficiency, a second round of capture was performed on the total amplified DNA from the first capture. Following the second round of capture, the library was quantified using KAPA library quantification kit using the average size calculated from Bioanalyzer. Libraries were then sequenced on Illumina HiSeq-4000 (2x 150bp).
GEO Accession
GSM4314848