Please use this identifier to cite or link to this item:
Title: Chromatin interaction analysis with Paired-End Tag Sequencing (ChIA-PET) for mapping chromatin Interactions and Understanding Transcription Regulation
Authors: Goh, Y.
Fullwood, M.J.
Poh, H.M.
Peh, S.Q.
Ong, C.T.
Zhang, J.
Ruan, X.
Ruan, Y. 
Keywords: ChIA-PET
Chromatin Interactions
Issue 62
Next-Generation Sequencing
Issue Date: 30-Apr-2012
Citation: Goh, Y., Fullwood, M.J., Poh, H.M., Peh, S.Q., Ong, C.T., Zhang, J., Ruan, X., Ruan, Y. (2012-04-30). Chromatin interaction analysis with Paired-End Tag Sequencing (ChIA-PET) for mapping chromatin Interactions and Understanding Transcription Regulation. Journal of Visualized Experiments (62) : -. ScholarBank@NUS Repository.
Abstract: Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces. Such architectures are not random and involve interactions between gene promoters and regulatory elements. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures. 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. 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. © 2012 Creative Commons Attribution License.
Source Title: Journal of Visualized Experiments
ISSN: 1940087X
DOI: 10.3791/3770
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.


checked on Aug 4, 2020


checked on Jul 27, 2020

Page view(s)

checked on Aug 1, 2020

Google ScholarTM



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.