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.