UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyProgram in Systems Biology
Document Type
Journal ArticlePublication Date
2018-08-01Keywords
Biochemistry, Biophysics, and Structural BiologyGenetics and Genomics
Laboratory and Basic Science Research
Systems Biology
Metadata
Show full item recordAbstract
This protocol, which describes how 3C libraries are made from formaldehyde-fixed mammalian cells, starts with cells that were previously cross-linked. Before embarking on a 3C-based analysis, decide which restriction enzyme will be used. Two parameters must be considered. First, the frequency with which a restriction enzyme cuts will determine the size of the restriction fragments and thus the resolution with which chromatin interactions can be detected. In most studies, researchers use restriction enzymes that cut once every approximately 4 kb. If the intention is to analyze the composition of the 3C library using the 4C method, then a frequently cutting restriction enzyme that has a recognition sequence of four bases should be chosen. This is to ensure efficient inverse PCR-mediated amplification of ligation fragments. Second, using a restriction enzyme that cuts between elements of interest will result in each being assigned to a different restriction fragment. In that case, interactions can be studied for each individual genomic element of interest. It is not recommended to use restriction enzymes at temperatures higher than 37 degrees C because prolonged incubation at higher temperatures will result in reversal of formaldehyde-induced cross-links. Finally, to avoid biased digestion of differentially methylated DNA, use a restriction enzyme that is not sensitive to DNA methylation.Source
Cold Spring Harb Protoc. 2018 Aug 1;2018(8):pdb.prot097840. doi: 10.1101/pdb.prot097840. Link to article on publisher's site
DOI
10.1101/pdb.prot097840Permanent Link to this Item
http://hdl.handle.net/20.500.14038/49866PubMed ID
30068590Related Resources
ae974a485f413a2113503eed53cd6c53
10.1101/pdb.prot097840