Active Motif's ChIP-exonuclease (ChIP-exo) Kit offers an optimized and streamlined protocol for high-resolution genome-wide mapping of transcription factor binding sites in the human and mouse genomes. ChIP-exo is a modified ChIP-Seq approach that enables researchers to identify protein-DNA binding sites at a resolution of 20-95 base pairs, providing the ability to more precisely define DNA motifs and refine genome-wide protein binding profiles for studies of mutational or SNP effects on transcription factor binding sites.
Technology covered under U.S. Patent No. 8367334 b2.
ChIP-exo improves resolution of protein-DNA binding sites.
- Identifies transcription factor binding sites at 20-95 bp resolution compared to traditional ChIP-Seq resolution of ≥200 bp
- Enables identification of unique transcription factor binding sites not revealed by ChIP-Seq
- Produces lower background and requires lower depth of coverage due to higher resolution of reads
- Requires less cells to produce comparable resolution as published methods (increasing cell numbers will increase the number of identified peaks)
- On-bead enzymatic reactions streamline sample processing
- Go from cells to sequence-ready library in a single assay
For analysis of data, we recommend using a program designed to evaluate ChIP-exo called MACE (Model-Based Analysis of ChIP-exo). MACE will look at the border peaks on either side of the binding site to generate very concise sequencing information. For details on this program, or to download the software please visit
To learn more about Active Motif's ChIP-exo assay, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.
|ChIP-exo Kit||12 rxns||53043||$1,865||Buy Now|
|High Sensitivity Chromatin Preparation||16 rxns||53046||$255||Buy Now|
|Indexing Primer 2||6 rxns||53090||$45||Buy Now|
|Indexing Primer 4||6 rxns||53091||$45||Buy Now|
|Indexing Primer 5||6 rxns||53092||$45||Buy Now|
|Indexing Primer 6||6 rxns||53093||$45||Buy Now|
|Indexing Primer 7||6 rxns||53094||$45||Buy Now|
|Indexing Primer 12||6 rxns||53095||$45||Buy Now|
|Indexing Primer 13||6 rxns||53096||$45||Buy Now|
|Indexing Primer 14||6 rxns||53097||$45||Buy Now|
|Indexing Primer 15||6 rxns||53098||$45||Buy Now|
|Indexing Primer 16||6 rxns||53099||$45||Buy Now|
|Indexing Primer 18||6 rxns||53088||$45||Buy Now|
|Indexing Primer 19||6 rxns||53089||$45||Buy Now|
How does Active Motif's ChIP-exo Assay work?
Transcriptional regulation results from the highly dynamic interactions between epigenetic modifications, transcription factors and cofactors, and chromatin. Aberrant transcription is often at the root of various human diseases, including cancer. Therefore, gaining further insight into the mechanisms regulating gene expression is crucial to understanding disease susceptibility, initiation and progression. ChIP-Sequencing (ChIP-Seq) and ChIP-chip are commonly utilized methods for analyzing DNA/protein interactions across the genome to provide insight into gene regulation. However, ChIP-Seq and ChIP-chip methods provide limited resolution and can have high background, limiting sequence coverage and increasing noise.
To overcome these limitations of ChIP-Seq and ChIP-chip, Active Motif's ChIP-exo method offers researchers a way to highly improve the resolution of protein-DNA binding sites. ChIP-exo is a modified ChIP-Seq approach that utilizes exonuclease digestion to reduce DNA fragments to the site of transcription factor binding prior to deep sequencing. The advantage of ChIP-exo is that it generates superior resolution of DNA binding protein maps by decreasing the number of reads mapping outside of peaks and increasing the accuracy of identification of protein-DNA binding motifs. The method is also more effective at detecting transcription factors that are inefficiently bound to the genome. With Active Motif’s ChIP-exo Kit, transcription factor binding site resolution is significantly improved, with ranges in length from 20-95 bp as compared to traditional ChIP-Seq resolution of 200 bp or more.
Figure 1: Schematic of Active Motif's ChIP-exo Assay.
The ChIP-exonuclease (ChIP-exo) assay utilizes exonuclease to digest back DNA to the site of transcription factor binding during chromatin immunoprecipitation to reduce background and significantly improve the resolution of transcription factor binding locations. Unlike ChIP-Seq methods that resolve of binding sites of 200 bp or more, the ChIP-exo method achieves resolution of protein binding sites within 20-95 bp to enable more precision in defining DNA motifs to facilitate studies of mutational or SNP effects.
ChIP-exonuclease Assay Advantages
- Achieves high-resolution genome-wide mapping of transcription factor binding sites using smaller amounts of cells than published ChIP-exo methods
- Enables identification of unique binding sites undetectable by ChIP-Seq
- Exonuclease-mediated reduction in DNA fragments prior to sequencing increases accuracy of protein-DNA binding motif identification
Active Motif’s ChIP-exo achieves comparable resolution of binding sites utilizing smaller amounts of cell numbers than previously published ChIP-exo methods
ChIP-exo was performed using Active Motif’s ChIP-exo method on chromatin fragments obtained from FoxA1 and CTCF ChIP experiments and compared to previously published ChIP-exo data for these transcription factors. These data shown below reveal Active Motif's ChIP-exo method is able to achieve comparable resolution of binding sites with smaller amounts of cells than published ChIP-exo methods.
Table 1: Table of results generated from bioinformatic analysis of ChIP-exo data of transcription factor FoxA1.
Figure 1: Comparison of Active Motif’s ChIP-exo data with published ChIP-exo data for FoxA1 shows similar resolution of FoxA1 binding sites is achievable using smaller cell amounts.
Table 2: Table of results generated from bioinformatic analysis of ChIP-exo data of transcription factor CTCF
Figure 2: Comparison of Active Motif’s ChIP-exo data with published ChIP-exo data for CTCF shows similar resolution of CTCF binding sites is achievable using smaller cell amounts.
ChIP-exo improves the detection of weak binding events, enabling identification of unique binding interactions with the genome that are not revealed by ChIP-Seq.
Figure 3: ChIP-exo identifies unique p53 binding sites within the p21 locus.
PCR walking primers can be developed to confirm the exonuclease digestion by ChIP-exo of the ChIP DNA prior to sequencing for known transcription factor binding sites.
Figure 4: PCR walking primers reveal that ChIP-exo reduces the size of the end ChIP chromatin product.
Contents & Storage
Please note that Active Motif's ChIP-exo Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Magnetic Beads after you have received this kit. This kit includes the following components:
- T4 Ligase (2000 U/µl); Store at -20°C
- DNA Polymerase I Klenow Fragment (5 U/µl); Store at -20°C
- T4 Polynucleotide Kinase (10 U/µl); Store at -20°C
- T4 DNA Polymerase (3 U/µl); Store at -20°C
- Phi29 Polymerase (10 U/µl); Store at -20°C
- Lambda Exonuclease (5 U/µl); Store at -20°C
- RecJf Exonuclease (30 U/µl); Store at -20°C
- Q5 High-Fidelity DNA Polymerase (2 U/µl); Store at -20°C
- 5X Q5 Reaction Buffer; Store at -20°C
- 1X Phi29 Reaction Buffer; Store at -20°C
- 10X T4 DNA Ligase Buffer; Store at -20°C
- 10X Lambda Exonuclease Buffer; Store at -20°C
- 10X Reaction Buffer AM3; Store at -20°C
- dNTPs (5 mM); Store at -20°C
- 100 mM ATP; Store at -20°C
- Indexing Primer 2; Store at -20°C
- Indexing Primer 4; Store at -20°C
- P7 exo-adapter; Store at -20°C
- P5 exo-adapter; Store at -20°C; Store at -20°C
- P7 Primer; Store at -20°C
- RNase A (10 µg/µl); Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Precipitation Buffer; Store at -20°C
- Carrier; Store at -20°C
- Glycogen; Store at -20°C
- Fixation Buffer; Store at 4°C
- Blocking Buffer AM3; Store at 4°C
- Protein G Magnetic Beads; Store at 4°C
- AMPure Beads; Store at 4°C
- 10X Wash Buffer AM5; Store at 4°C
- 10X Wash Buffer AM6; Store at 4°C
- Stop Solution; Store at RT
- Chromatin Prep Buffer; Store at RT
- ChIP Buffer; Store at RT
- Elution Buffer AM4; Store at RT
- DNA Purification Elution Buffer; Store at RT
- 5 M NaCl; Store at RT
- TE, pH 8.0; Store at RT
- Detergent; Store at RT
- Bar magnet (1 ea); Store at RT
- Glue dots (2 ea); Store at RT
The selected papers below cite the use of and/or provide additional information about ChIP-exo or relevant software analysis programs:
- “Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution” by Rhee, H.S. and Pugh, B.F. (2011) Cell 147(6):1408-1419.
- “Genome-wide Structure and Organization of Eukaryotic Pre-Initiation Complexes” by Rhee, H.S. and Pugh, B.F. et al (2012) Nature 483(7389):295-301; Erratum 487(7405):128.
- “Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties” by Serandour, A.A. et al (2013) Genome Biology 14(12):R147.
- “MACE: model based analysis of ChIP-exo” by Wang, L. et al (2014) Nucleic Acids Res. doi: 10.1093/nar/gku846.