GSM4624693: Capture-C-Clone21-CTCF&TSS-CRISPR#1-1st-rep2-seq; Homo sapiens; OTHER
Capture-C was performed as previously described (Hughes et al., Nat. Genet., 2014; Huang et al., Genes Dev., 2017; Davies et al., Nat. Methods, 2016). Briefly, ~10 million cells HAP1 cells were crosslinked in 1% formaldehyde at room temperature for 10 minutes, and then quenched in 1M glycine for 5 minutes, on an orbital shaker. Cells were then pelleted, washed with 1mL cold PBS, transferred to an Eppendorf tube, pelleted, resuspended in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), and incubated on ice for 10 minutes. Nuclei were then pelleted, washed once using NEBuffer™ DpnII, pelleted, and resuspended in 500µL NEBuffer™ DpnII. Samples were incubated in 0.3% SDS at 37°C, 950 rpm for 1 hour in a thermomixer, followed by the addition of a final concentration of 1.8% Triton X-100, for 1 hour at 37°C, 950 rpm. After adding 40µL water, 300U DpnII was added for in situ digestion at 37°C, overnight. An additional 300U DpnII was then added, and samples were incubated for 4 more hours at 37°C, followed by 20 minutes at 65°C, 950 rpm. Nuclei were pelleted, resuspended in a total volume of 1.2mL with 4000U T4 DNA Ligase (NEB), and incubated at 16°C for 4 hours and then at room temperature for 30 minutes. 20µL of 20mg/mL Proteinase K and 120µL of 10% SDS were added, and samples were incubated at 65°C overnight to reverse crosslinking. An additional 10µL proteinase K was added to each sample for 2 more hours at 55°C. 2µL DNase-free RNAase was added to each sample, followed by incubation at 37°C for 30 minutes. DNA was then extracted with phenol-chloroform. DNA was sonicated to 200-300bp in a bath sonicator (QSonica Q800R3), followed by beads clean-up. Sequencing libraries were subsequently prepared similarly as described in "Insertion-site-mapping” samples: End Repair, dA-Tailing, adaptor ligation, 6 cycles of indexing PCR, and 6 cycles of amplification using P5/P7. After sequencing library preparation, two rounds of target enrichment/capture were carried out using biotinylated hybridization oligos similarly as described in "Insertion-site-mapping” samples. Pulldown enrichment was confirmed using qPCR. Target enriched libraries were quality checked and quantified as in situ Hi-C samples prior to 2 x 75 bp sequencing on the Illumina NextSeq 500. See "extract protocol"