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ChIP-exo

アクティブ・モティフ社のChIP-exonuclease (ChIP-exo) キットは,ヒトおよびマウスゲノム中の転写因子結合部位を高解像度にゲノムワイドにマッピングするために最適化かつ合理化されたプロトコールを提供します。ChIP-exoは,転写因子結合部位での突然変異またはSNP研究のために,より正確にDNAモチーフを定め,ゲノムワイドなタンパク質結合プロファイルを発生させる能力を改良することで,タンパク質-DNA結合部位を20-95 bpの解像度で特定できるように改変されたChIP-Seqアプローチです。

Technology covered under U.S. Patent No. 8367334 b2.

ChIP-exo improves resolution of protein-DNA binding sites.
ChIP-exo はタンパク質-DNA結合部位の解像度を改善する。

ChIP-exo の利点

  • ≥200 bpの一般的なChIP-Seq解像度と比較して,20-95 bp解像度で転写因子結合部位を特定します。
  • ChIP-Seqによって明らかにされないユニークな転写因子結合部位の識別が可能です。
  • リード解像度が高いため,低バックグラウンドです。
  • 公開されている実験手法(細胞数の増加により同定されるピーク数は増加する)の通り,同程度の解像度を得るために多くの細胞を必要としません。
  • ビーズ上での酵素反応は,試料処理を合理化します。
  • 1回のアッセイで,細胞からシーケンスできるよう準備されたライブラリー作製までを行えます。

データ解析のために, MACE (Model-Based Analysis of ChIP-exo) と呼ばれるChIP-exoを評価するためプログラムを使用することを推奨します。 MACEは,非常に簡潔な配列情報を生み出すために,結合部位の両サイドの境界ピークを調べます。このプログラムの詳細またはソフトウェアをダウンロードするために http://dldcc-web.brc.bcm.edu/lilab/MACE/docs/html/ をご覧ください。

アクティブ・モティフ社のChIP-exo assayの詳細については,以下のMethodData または Contents タブをクリックしてください。 マニュアルまたは関連書類については,Documents タブをクリックしてください。

 
Name Format Cat No. 価格 (税抜)  
ChIP-exo Kit 12 rxns 53043 ¥350,000 Buy Now
High Sensitivity Chromatin Preparation 16 rxns 53046 ¥47,000 Buy Now
Indexing Primer 2 6 rxns 53090 ¥8,800 Buy Now
Indexing Primer 4 6 rxns 53091 ¥8,800 Buy Now
Indexing Primer 5 6 rxns 53092 ¥8,800 Buy Now
Indexing Primer 6 6 rxns 53093 ¥8,800 Buy Now
Indexing Primer 7 6 rxns 53094 ¥8,800 Buy Now
Indexing Primer 12 6 rxns 53095 ¥8,800 Buy Now
Indexing Primer 13 6 rxns 53096 ¥8,800 Buy Now
Indexing Primer 14 6 rxns 53097 ¥8,800 Buy Now
Indexing Primer 15 6 rxns 53098 ¥8,800 Buy Now
Indexing Primer 16 6 rxns 53099 ¥8,800 Buy Now
Indexing Primer 18 6 rxns 53088 ¥8,800 Buy Now
Indexing Primer 19 6 rxns 53089 ¥8,800 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.

Schematic of Active Motif's ChIP-exo method
Figure 1: Schematic of Active Motif's ChIP-exo Assay.

In Active Motif's ChIP-exo method, cells are fixed with formaldehyde to cross-link protein-DNA binding interactions. The cells are then lysed and chromatin is fragmented by sonication. An antibody directed against the protein of interest is conjugated to protein G magnetic beads for immunoprecipitation of the DNA of interest. Following chromatin immunoprecipitation, with chromatin still bound to the beads, the DNA is end-polished and P7-exo adaptor sequences are ligated to the ChIP DNA fragments to demarcate 3´ ends prior to 5´ end exonuclease digestion. The nicked DNA is repaired and then digested by lambda and RecJF exonucleases to excise DNA flanking the site of DNA-protein binding and eliminate the 5´ P7 adaptor sequence of each strand. DNA is eluted from the beads, cross-linking is reversed and DNA fragments are denatured. The DNA is then made double-stranded by primer extension followed by ligation of a second set of sequencing adaptors (P5-exo) to the exonuclease-treated ends. The DNA library is PCR amplified and size selected before it is subjected to high-throughput sequencing. The sequence of the DNA fragments is mapped back to the reference genome to determine the binding locations of the protein of interest. The 5´ ends of DNA fragments on the forward strand indicate the left border of a DNA-protein interaction, while the 5´ ends of DNA fragments on the reverse strand indicate the right border of a DNA-protein interaction. These borders demarcate the precise site of the protein-DNA cross-linking, providing high-resolution (20-95 base pairs) identification of genomic binding locations compared to ChIP-chip and ChIP-Seq technologies that can only achieve a resolution of 200 base pairs or more in length of transcription factor bound DNA motifs.

ChIP-exonuclease 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.


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.

Example of data analysis from ChIP-exo with transcription factor FoxA1.
Table 1: Table of results generated from bioinformatic analysis of ChIP-exo data of transcription factor FoxA1.

Active Motif’s ChIP-exo Kit was used to analyze transcription factor FoxA1 for comparison to published data. Bioinformatic analysis was performed using both MACS and MACE programs. The data displays comparable results between the kit and published results. The identified FoxA1 binding sites are significantly smaller for ChIP-exo than traditional ChIP-Seq results. This data shows the reproducibility of the assay for binding site identification.

BigWig graphs and Venn diagrams comparing Active Motif’s ChIP-exo data with published ChIP-exo data for 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.

To generate Active Motif’s ChIP-exo results (AM) for FoxA1, 100 μg of chromatin obtained from 20 million MCF-7 cells was fragmented and ChIP was performed using the ChIP-exo Kit with antibodies against FoxA1. Results were compared with published results (Published) for FoxA1 obtained by performing ChIP-exo on fragmented chromatin generated from 100 million MCF-7 cells as described previously by Serandour et al. (2013) Genome Biol. 14:R147. Data was aligned to the hg19 human reference genome using Bowtie. The BigWig graphs were generated using the ChIP-exo-specific analysis software: MACE (Model based Analysis of ChIP-exo). Results overlap with reported FoxA1 binding sites. Venn diagrams are also depicted showing the number of unique and overlapping peaks generated by FoxA1 ChIP-exo and ChIP-Seq experiments along with the most frequently detected FoxA1 binding motifs by ChIP-exo analysis. These results demonstrate that Active Motif’s ChIP-exo method achieves comparable resolution of binding motifs as the published data for FoxA1 using a fraction of the starting cell numbers used to obtain the published data.

Example of data analysis from ChIP-exo with transcription factor CTCF.
Table 2: Table of results generated from bioinformatic analysis of ChIP-exo data of transcription factor CTCF

Active Motif’s ChIP-exo Kit was used to analyze transcription factor CTCF for comparison to published data. Bioinformatic analysis was performed using both MACS and MACE programs. The data displays comparable results between the kit and published results. The identified CTCF binding sites are significantly smaller for ChIP-exo than traditional ChIP-Seq results. This data shows the reproducibility of the assay for binding site identification.

BigWig graphs and Venn diagrams comparing Active Motif’s ChIP-exo data with published ChIP-exo data for 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.

To generate Active Motif’s ChIP-exo results (AM) for CTCF, 100 μg of chromatin obtained from 20 million HeLa cells was fragmented and ChIP was performed using the ChIP-exo Kit with antibodies against CTCF. Results were compared with published results (Published) for CTCF obtained by performing ChIP-exo on fragmented chromatin generated from 60 million HeLa cells as described previously by Rhee et al. (2011) Cell, 147(6):1408-1419. Data was aligned to the hg19 human reference genome using Bowtie. The BigWig graphs were generated using the ChIP-exo-specific analysis software: MACE (Model based Analysis of ChIP-exo). Results overlap with reported CTCF binding sites. Venn diagrams are also depicted showing the number of unique and overlapping peaks generated by CTCF ChIP-exo and ChIP-Seq experiments along with the most frequently detected CTCF binding motifs by ChIP-exo analysis. These results demonstrate that Active Motif’s ChIP-exo method achieves comparable resolution of binding motifs as the published data for CTCF using a fraction of the starting cell numbers used to obtain the published data.


ChIP-exo enables identification of unique binding sites

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.

ChIP-exo identifies less pronounced p53 binding sites within the p21 locus.
Figure 3: ChIP-exo identifies unique p53 binding sites within the p21 locus.

In addition to reducing the size of the identified p53 binding site (red triangle) as compared to ChIP-Seq results, Active Motif’s ChIP-exo method identifies several other unique binding sites not revealed by ChIP-Seq (red circles).


ChIP-exo reduces the size of the end ChIP chromatin product

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.

ChIP-exo reduces the size of the end ChIP chromatin product.
Figure 4: PCR walking primers reveal that ChIP-exo reduces the size of the end ChIP chromatin product.

Walking primers extending from the PLK2:p53 and SCN4A:CTCF binding sites indicate that the end chromatin fragment size is reduced by ChIP-exo (ChIP-exo) compared to fragmented chromatin without ChIP-exo processing (w/o ChIP-exo). MW = Molecular weight markers.

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: