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Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for mapping chromatin interactions and understanding transcription regulation.
Goh, Yufen; Fullwood, Melissa J; Poh, Huay Mei; Peh, Su Qin; Ong, Chin Thing; Zhang, Jingyao; Ruan, Xiaoan; Ruan, Yijun.
Afiliação
  • Goh Y; Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore.
J Vis Exp ; (62)2012 Apr 30.
Article em En | MEDLINE | ID: mdl-22564980
ABSTRACT
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces (7, 2, 12). Such architectures are not random and involve interactions between gene promoters and regulatory elements (13). The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination (1, 14). Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures (5,6). Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions (8). We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Assuntos

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Transcrição Gênica / Cromatina / Regulação da Expressão Gênica / Análise de Sequência de DNA / Imunoprecipitação da Cromatina Tipo de estudo: Prognostic_studies Idioma: En Revista: J Vis Exp Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Singapura

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Transcrição Gênica / Cromatina / Regulação da Expressão Gênica / Análise de Sequência de DNA / Imunoprecipitação da Cromatina Tipo de estudo: Prognostic_studies Idioma: En Revista: J Vis Exp Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Singapura