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samples were fixed with a final concentration of 1% formaldehyde and quenched with 0.125M glycine. Cells were lysed in ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, 1x protease inhibitor cocktail) for 15 min. Pelleted nuclei were washed once with 1x NEBuffer 2 and incubated in 0.5% sodium dodecyl sulfate (SDS) at 62°C for 5 min. After incubating, water and Triton X-100 were added to quench the SDS. MboI restriction enzyme (NEB, R0147) were then added and chromatin was digested. Biotin-14-dATP was used to mark the DNA ends followed by proximity ligation in intact nuclei. After crosslink reversal, samples were sheared to a length of 300 bp, then treated with the End Repair/dA-Tailing Module (NEB, E7442L) and Ligation Module (NEB, E7445L) following the operation manual. Biotin-labeled fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). The Hi-C library was amplified for about 11 cycles of PCR with Q5 master mix (NEB, M0492L) following the operation manual. DNA was then purified with size selection, quantified and sequenced from both ends using an Illumina sequencing platform. |
Hi-C |
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PCR |
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