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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| ChIP-exo | 1/2 | https://en.wikipedia.org/wiki/ChIP-exo | reference | science, encyclopedia | 2026-05-05T14:02:10.121307+00:00 | kb-cron |
ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest (transcription factor) binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.
== Theory == Chromatin immunoprecipitation (ChIP) techniques have been in use since 1984 to detect protein-DNA interactions. There have been many variations on ChIP to improve the quality of results. One such improvement, ChIP-on-chip (ChIP-chip), combines ChIP with microarray technology. This technique has limited sensitivity and specificity, especially in vivo where microarrays are constrained by thousands of proteins present in the nuclear compartment, resulting in a high rate of false positives. Next came ChIP-sequencing (ChIP-seq), which combines ChIP with high-throughput sequencing. However, the heterogeneous nature of sheared DNA fragments maps binding sites to within ±300 base pairs, limiting specificity. Secondly, contaminating DNA presents a grave problem since so few genetic loci are cross-linked to the protein of interest, making any non-specific genomic DNA a significant source of background noise. To address these problems, Rhee and Pugh revised the classic nuclease protection assay to develop ChIP-exo. This new ChIP technique relies on a lambda exonuclease that degrades only, and all, unbound double-stranded DNA in the 5′ to 3′ direction.
== Workflow ==
=== ChIP === Cells are crosslinked in vivo with formaldehyde to covalently bind proteins to DNA at their natural binding locations across a genome. Cells are then collected, broken open, and the chromatin sheared and solubilized by sonication. An antibody is then used to immunoprecipitate the protein of interest (engineering cells with an epitope tag can be useful for immunoprecipitation), along with the crosslinked DNA. DNA PCR adaptors are then ligated to the ends, which serve as a priming point for second strand DNA synthesis after the exonuclease digestion. Lambda exonuclease then digests double DNA strands from the 5′ end until digestion is blocked at the border of the protein-DNA covalent interaction. Most contaminating DNA is degraded by the addition of a second single-strand specific exonuclease. After the cross-linking is reversed, the primers to the PCR adaptors are extended to form double stranded DNA, and a second adaptor is ligated to 5′ ends to demarcate the precise location of exonuclease digestion cessation. The library is then amplified by PCR, and the products are identified by high throughput sequencing. This method allows for resolution of up to a single base pair for any protein binding site within any genome, which is a much higher resolution than either ChIP-chip or ChIP-seq.
=== Sequencing === ChIP-exo utilizes short read (e.g. Illumina NGS) sequencing. Sequencing requirements are lower for ChIP-exo than that of other assays like ChIP-seq because the dramatically reduced "shouldering" of a higher resolution assay like ChIP-exo means that the sampling of DNA fragments for constructing the DNA library are better dominated by target-bound sites (this effect can vary across different targets). For paired end data from a standard ChIP-exo prep, the 5' end of Read 1 sequenced from the DNA fragments marks the position of the cross-linking site (lambda exonuclease digestion stop site). Paired-end sequencing improves the mappability and specificity of read alignments, especially for large genomes.
== Protocols ==
=== ChIP-exo 1.x === ChIP-exo 1.x improves on ChIP-seq by generating data with higher positional resolution by adding a lambda exonuclease digestion step. This higher resolution enables the capture of the organization of factors within a complex. Where version 1.0 was originally designed for the ABI SOLiD platform, ChIP-exo 1.1 makes the assay compatible with the Illumina NGS platform.
=== ChIP-exo 2.x (ChIP-nexus) === ChIP-nexus utilizes a circular rather than linear DNA library, and increases efficiency of adapter ligation through CircLigase. However, the assay requires additional endonuclease digestion, and published ChIP-nexus data reports data loss due to poor barcode quality.
=== ChIP-exo 3.x === ChIP-exo 3.x employs one-step adapter attachment using Tn5 tagmentation. This version of ChIP uses fewer steps than previous protocols while simultaneously retaining high resolution. However, the libraries produced may be enriched for longer fragments, since tagmentation by Tn5 may occur at a higher frequency for such fragments.
=== ChIP-exo 4.x === ChIP-exo 4.x aims to streamline library construction and avoid the library biases of Tn5. ssDNA splint ligation is incorporated into the workflow. ChIP-exo 4.x is the simplest ChIP-exo version, but 4.0 may produce some steric exclusion of the adapter,and 4.1 may have lower precision.
=== ChIP-exo 5.0 === ChIP-exo 5.0 was developed to improve precision by reducing the "shouldering" found in versions 3.x and 4.x. Enzymatic steps are largely reduced, and as a result, library yield is greatly increased and signal concentration is maximized. 5.0 offers what the authors considered the best compromise in achieving high precision with a streamlined protocol at the time of publication.