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    Generation of 3C Libraries from Cross-Linked Cells

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    Authors
    Kim, Tae Hoon
    Dekker, Job
    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Program in Systems Biology
    Document Type
    Journal Article
    Publication Date
    2018-08-01
    Keywords
    Biochemistry, Biophysics, and Structural Biology
    Genetics and Genomics
    Laboratory and Basic Science Research
    Systems Biology
    
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    Link to Full Text
    https://doi.org/10.1101/pdb.prot097840
    Abstract
    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.prot097840
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/49866
    PubMed ID
    30068590
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    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1101/pdb.prot097840
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