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Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C.
Kolovos, Petros; Brouwer, Rutger W W; Kockx, Christel E M; Lesnussa, Michael; Kepper, Nick; Zuin, Jessica; Imam, A M Ali; van de Werken, Harmen J G; Wendt, Kerstin S; Knoch, Tobias A; van IJcken, Wilfred F J; Grosveld, Frank.
Afiliación
  • Kolovos P; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Brouwer RWW; Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, the Netherlands.
  • Kockx CEM; Center for Biomics, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Lesnussa M; Center for Biomics, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Kepper N; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Zuin J; Chromatin Networks, BioQuant & German Cancer Research Center, Heidelberg, Germany.
  • Imam AMA; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • van de Werken HJG; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Wendt KS; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Knoch TA; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • van IJcken WFJ; Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.
  • Grosveld F; Center for Biomics, Erasmus Medical Centre, Rotterdam, the Netherlands.
Nat Protoc ; 13(3): 459-477, 2018 03.
Article en En | MEDLINE | ID: mdl-29419817
Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Análisis de Secuencia de ADN / Biología Computacional / Mapeo Físico de Cromosoma Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Nat Protoc Año: 2018 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Análisis de Secuencia de ADN / Biología Computacional / Mapeo Físico de Cromosoma Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Nat Protoc Año: 2018 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido