Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Generation of 3C Libraries from Cross-Linked Cells

(Protocol summary only for purposes of this preview site)

This protocol, which describes how 3C libraries are made from formaldehyde-fixed mammalian cells, starts with cells that were cross-linked as described in Protocol 1. Before embarking on a 3C-based analysis, decide which restriction enzyme will be used. Two parameters must be considered.

  • 1.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 4 kb. If the intention is to analyze the composition of the 3C library using the 4C method (Protocol 11), then a frequently cutting restriction enzyme that has a recognition sequence of 4 bases should be chosen. This is to ensure efficient inverse PCR-mediated amplification of ligation fragments.
  • 2.Using a restriction 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 37C 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.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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