Analysis of long-range chromatin interactions using Chromosome Conformation Capture.
Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.
Programs in Systems Biology and Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-0103, USA.
Natalia
N
Naumova
Emily M
EM
Smith
Ye
Y
Zhan
Job
J
Dekker
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D053837:DNA Cleavage; D011088:DNA Ligases / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004843:Epistasis, Genetic; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction
D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D005404:Fixatives; D005557:Formaldehyde; D004262:DNA Restriction Enzymes; D011088:DNA Ligases