Title

Generation of 3C Libraries from Cross-Linked Cells

UMMS Affiliation

Program in Systems Biology; Department of Biochemistry and Molecular Pharmacology

Publication Date

2018-08-01

Document Type

Article

Disciplines

Biochemistry, Biophysics, and Structural Biology | Genetics and Genomics | Laboratory and Basic Science Research | Systems Biology

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.

DOI of Published Version

10.1101/pdb.prot097840

Source

Cold Spring Harb Protoc. 2018 Aug 1;2018(8):pdb.prot097840. doi: 10.1101/pdb.prot097840. Link to article on publisher's site

Journal/Book/Conference Title

Cold Spring Harbor protocols

Related Resources

Link to Article in PubMed

PubMed ID

30068590

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