Authors
Phillips Y.H. Huang, Yuyuan Han, Lusy Handoko, Stoyan Velkov, Eleanor Wong, Edwin Cheung, Xiaoan Ruan, Chia-Lin Wei, Melissa Jane Fullwood, and Yijun Ruan
Summary
The three-dimensional organization of chromatin in the nuclear space is involved in regulation of gene expression. Circular Chromosome Conformation Capture (4C) is an established method for genome-wide screening of chromatin interactions associated with a given locus of interest, without prior knowledge of the identity of interacting partners. Briefly, 4C involves the cross-linking of chromatin material, followed by restriction enzyme digestion of the chromatin, proximity-based ligation of interacting DNA fragments within the same DNA-protein complex, and amplification of interacting sequences by inverse PCR. The use of restriction enzyme digestion together with PCR could lead to biases in detection as well as false positives through repeated identification of clonal products. Here, we present a modification of the original 4C method, in which sonication is used to randomly fragment chromatin fibers instead of restriction enzymes at specific sites, to eliminate these biases, thus enabling high-throughput analysis by next-generation sequencing to detect interacting sequences.Further details
The protocol was published on Protocol Exchange on 14 December 2010. To see the entire protocol, click on the source link.Advertisement
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