Journal
NATURE PROTOCOLS
Volume 2, Issue 4, Pages 988-1002Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.116
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Funding
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [R01HG003143] Funding Source: NIH RePORTER
- NHGRI NIH HHS [HG003143] Funding Source: Medline
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Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.
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