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Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment., (© 2015 Cold Spring Harbor Laboratory Press.)

Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes., (© 2015 Cold Spring Harbor Laboratory Press.)

Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing., (© 2015 Cold Spring Harbor Laboratory Press.)

Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR)., (© 2015 Cold Spring Harbor Laboratory Press.)