Unbiased interrogation of 3D genome topology using chromosome conformation capture coupled to high-throughput sequencing (4C-Seq)

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• dc.contributor.author Brouwer, Rutger W.ca
• dc.contributor.author Hout, Mirjam C. van denca
• dc.contributor.author IJcken, Wilfred F. vanca
• dc.contributor.author Soler, Ericca
• dc.contributor.author Stadhouders, Ralphca
• dc.date.accessioned 2017-11-16T08:33:06Z
• dc.date.available 2017-11-16T08:33:06Z
• dc.date.issued 2017
• dc.description.abstract The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.
• dc.format.mimetype application/pdfca
• dc.identifier.citation Brouwer RW, van den Hout MC, van IJcken WF, Soler E, Stadhouders R. Unbiased interrogation of 3D genome topology using chromosome conformation capture coupled to high-throughput sequencing (4C-Seq). Methods Mol Biol. 2017; 1507: 199-220. DOI: 10.1007/978-1-4939-6518-2_15
• dc.identifier.doi http://dx.doi.org/10.1007/978-1-4939-6518-2_15
• dc.identifier.issn 1064-3745
• dc.identifier.uri http://hdl.handle.net/10230/33249
• dc.language.iso eng
• dc.publisher Humana Press (Springer Imprint)ca
• dc.relation.ispartof Methods in Molecular Biology. 2017;1507:199-220
• dc.rights © Springer The final publication is available at Springer via http://dx.doi.org/10.1007/978-1-4939-6518-2_15
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.subject.other Genòmica
• dc.subject.other Bioinformàtica
• dc.subject.other ADN
• dc.subject.other Cromatina
• dc.subject.other Cromosomes
• dc.title Unbiased interrogation of 3D genome topology using chromosome conformation capture coupled to high-throughput sequencing (4C-Seq)ca
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/acceptedVersion