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DNA and protein were cross-linked by formaldehyde, NP-40 cleavage, restriction endonuclease DPN II digestion, terminal repair, biotin-14-dCTP labeling and performing blunt-end ligation of crosslinked fragments. Proteinase K digestion removed protein, released DNA, and DNA was purified by phenol-chloroform extraction. Biotin-C was removed from non-ligated fragment ends using T4 DNA polymerase. Fragments were sheared to a size of 200-600 basepairs by sonication. The fragment ends were repaired by the mixture of T4 DNA polymerase, T4 polynucleotide kinase and Klenow DNA polymerase. Biotin labeled HiC sample were specifically enriched using streptavidin C1 magnetic beads. The fragment ends were adding A-tailing and then adding Illumina paired-end sequencing adapter by ligation mix. At last, the Hi-C libraries were amplified by 12-14 cycles PCR , and sequenced in Illumina HiSeq platform. |
Hi-C |
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PCR |
PAIRED
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