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TAM-ChIP

1ステップでゲノムターゲティングとライブラリー生成が可能

Active Motif's TAM-ChIP* technology is a robust chromatin immunoprecipitation (ChIP) method that combines the power of Nextera** next-generation library preparation with ChIP for directed genomic targeting and library generation in one. TAM-ChIP utilizes the specificity of a ChIP-seq validated antibody to target protein binding sites of interest and a TAM-ChIP anti-species antibody conjugate in combination with a unique TsTn5 transposase to deliver barcoded adapter sequences directly into the chromatin surrounding the binding site. These adapter sequences are necessary for library amplification and sequencing using Illumina® platforms. By incorporating the library preparation into the ChIP reaction, you avoid sample loss commonly associated with the multiple end-repair and ligation steps of traditional library preparation methods.

    *Patent pending
    **Nextera is a trademark of Epicentre, Madison, Wisconsin.

TAM-ChIP Highlights

  • Tagmentation of the DNA using the TsTn5 transposase aids in chromatin fragmentation and provides higher resolution of protein binding sites
  • Avoid sample loss common with traditional library preparation steps
  • Highly robust procedure has been validated to profile both histone and transcription factor DNA binding sites
  • Molecular identifiers (8 bp random barcodes) enable distinction of PCR duplicates from biological replicates during bioinformatic analysis to increase the number of unique alignments 3-fold
  • Multiplex up to 16 samples on the same sequencing flow cell

 

Video demonstrating the TAM-ChIP technology.

 

To perform TAM-ChIP you will need a ChIP-validated antibody and the appropriate anti-species TAM-ChIP conjugate. Validated TAM-ChIP Assay Reagents are also available. To learn more about TAM-ChIP, please select the Method or Data tabs below. To download a copy of the manual or the data sheets, please select the Documents tab.

 
Name Format Cat No. 価格 (税抜)  
TAM-ChIP anti-rabbit conjugate 16 rxns 53126 ¥172,000 Buy Now
TAM-ChIP anti-mouse conjugate 16 rxns 53127 ¥172,000 Buy Now
TAM-ChIP Assay Reagents 16 rxns 53128 ¥93,000 Buy Now

How does TAM-ChIP work?

TAM-ChIP is designed to perform antibody-directed genomic targeting and NGS library preparation at once. First, intact cells are fixed with formaldehyde which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication and incubated with an antibody directed against the DNA-binding protein of interest. Then, a species-specific TAM-ChIP antibody conjugate containing Illumina-compatible sequencing adapters is added to bind the ChIP antibody. Activation of the unique TsTn5 transposase cuts the nearby DNA surrounding the genomic region of interest and pastes the antibody-associated adapters into the DNA sequence. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed to remove non-specific DNA. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K, and the DNA is recovered and purified. ChIP enriched DNA is now ready for library amplification and sequencing.

TAM-ChIP combines antibody directed protein targeting and library generation

By combining the next-generation library preparation within the ChIP reaction, TAM-ChIP minimizes sample loss. Traditional methods to prepare ChIP-Seq libraries involve a series of processing steps to end repair the DNA and ligate sequencing adapters. This multi-step process can often lead to significant loss of sample material. Another advantage of TAM-ChIP is the inclusion of random barcodes in the adapter sequences. This enables distinction of PCR duplicates from biological replicates during bioinformatic analysis to increase the number of unique alignments.

 

The TAM-ChIP Flowchart

TAM-ChIP is a multi-step process that involves assembling the unique TsTn5 transposase to the secondary antibody conjugate and an oligonucleotide, prior to immunoprecipitation (A). The TAM-ChIP anti-species antibody conjugate contains a single-stranded oligonucleotide comprised of the i5 antibody index for Next-generation sequencing. Each anti-species antibody has its own i5 index to enable multiplexing of both the anti-rabbit and anti-mouse TAM-ChIP conjugates within the same sequencing reaction. When the antibody conjugate is combined with a single-stranded pMENTS oligonucleotide, the complementary sequences anneal to create a double-stranded recognition sequence for the TsTn5 transposase to assemble. This process loads the inactivated transposase onto the antibody conjugate. At the same time, a single stranded oligonucleotide is also annealed to pMENTS and loaded with inactivated transposase.

During immunoprecipitation (B), the ChIP-validated antibody of interest is combined with sonicated chromatin and incubated overnight. The next day, the assembled TAM-ChIP antibody conjugate is added to the immunoprecipitation reaction. Following a one hour incubation, protein G agarose beads are added to capture the chromatin fragments of interest. Each ChIP reaction is then loaded onto a ChIP filtration column and the oligonucleotide assembled with transposase is added to the column. The TsTn5 transposase is then activated to insert the i5 antibody index and oligonucleotide sequence into the genomic region surrounding the protein of interest. Following washing, elution, Proteinase K treatment, reversal of cross-links and DNA purification, the ChIP library is ready for amplification.

The TAM-ChIP Library is amplified by PCR prior to sequencing (C). During PCR, the second strand of the DNA template is extended. Then, the i7 TAM-ChIP Index which contains the 3 bp sample barcode and 8 bp random molecular identifier (MID) for PCR de-duplication is incorporated into the template. Additional PCR cycles amplify the entire template using the amplification primer mix. Libraries are size-selected and sequenced using Illumina® platforms.

 

Overview of the TAM-ChIP workflow

TAM-ChIPはどのように機能するか?

 TAM-ChIPは、抗体指示のゲノムターゲティングおよびNGSライブラリー調製を一度に行うように設計されています。 最初に、タンパク質/ DNA相互作用をクロスリンクして保存するために、無傷細胞をホルムアルデヒドで固定します。 次いで、DNAをソニケーションで小さな断片にせん断し、目的のDNA結合タンパク質に対する抗体とインキュベートします。 それから、Illumina互換性シーケンシングアダプターを含む種特異的TAM-ChIP抗体コンジュゲートを加えてChIP抗体を結合させます。 独特なTsTn5トランスポゼースの活性化は、目的のゲノム領域周辺近くのDNAを切断し、抗体結合アダプターをDNA配列にペーストします。 抗体結合タンパク質/ DNA複合体をプロテインGアガロースビーズの使用により免疫沈降させ、洗浄して非特異的DNAを除去します。 免疫沈降の後、脱クロスリンク、Proteinase Kによりタンパク質を除去、DNAを回収し精製します。 これで、ライブラリー増幅とシーケンシングのためのDNAが濃縮されたChIPサンプルの準備が整いました。

ChIP反応で次世代ライブラリー調製を組み合わせることにより、TAM-ChIPはサンプルの損失を最小限に抑えます。 ChIP-Seqライブラリーを調製する従来の方法では、DNAの末端修復およびシーケンシングアダプターの連結をするための一連の処理工程を行います。 この多段階過程は、しばしばサンプル物質のかなりな損失を招くことがあります。 TAM-ChIPの他の利点は、アダプター配列にランダムなバーコードを含めることです。 これにより、バイオインフォマティクス解析中のPCR重複物と生物学的複製との区別が可能になり、ユニークなアラインメントの数が増加します。

 

TAM-ChIP combines antibody directed protein targeting and library generation

 


頑健で再現性の高いデータ

 アクティブ・モティフのTAM-ChIPアッセイ試薬を使用して、MCF-7細胞からの10μgのクロマチンでTAM-ChIPを実施した。インシュレータータンパク質であるCTCF4μg)およびHistone H3K4me34μg)を標的とする抗体を4μgTAM-ChIP抗ウサギコンジュゲートと組み合わせて使用した。PCRでライブラリーを増幅し、Illumina NextSeq 500を用いてシーケンスをした.ChIP-Seqピークを、UCSCゲノムブラウザ上で利用可能なENCODEデータセットと比較した。結果は、公開されたデータセットと一致する複製物とピークとの間の再現性を示します。

TAM-ChIP data for CTCF and Histone H3K4me3.
1ENCODEデータセットと比較した、CTCFおよびヒストンH3K4me3TAM-CHIPシーケンシングピーク。

 


 

 

TAM-ChIPで検証された抗体

さらに標的タンパク質が豊富に含まれるかおよびChIP-Seq抗体の品質どうかがTAM-ChIPの成功に影響を与える要因となります。 以下に、TAM-ChIPで使用可能なActive Motifの有効な抗体を紹介します。

Protein Target Active Motif Catalog Number Cell Type Tested
H3K4me3 39915 MCF-7
H3K9ac 39917 MCF-7
H3K27ac 39685 MCF-7
CTCF 61311 MCF-7
RNA pol II 39097 MCF-7
1TAM-ChIPで検証された抗体の表。

 

Contents & Storage

Please note that the TAM-ChIP Assay Reagents are shipped on dry ice and contains reagents with multiple storage temperatures inside. All components can be stored at -20°C prior to first use, then we recommend storing each component at the temperatures indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Each kit includes the following components:

Reagents for TAM-ChIP anti-rabbit conjugate

  • TAM-ChIP anti-rabbit antibody conjugate; Store at 4°C
  • pMENTS AR; Store at -20°C
  • Oligonucleotide; Store at -20°C
  • TsTn5 transposase; Store at -20°C
  • 5X Tagmentation Buffer; Store at -20°C
  • TAM Index 1; Store at -20°C
  • TAM Index 2; Store at -20°C
  • TAM Index 3; Store at -20°C
  • TAM Index 4; Store at -20°C
  • TAM Index 5; Store at -20°C
  • TAM Index 6; Store at -20°C
  • TAM Index 7; Store at -20°C
  • TAM Index 8; Store at -20°C
  • TAM Index 9; Store at -20°C
  • TAM Index 10; Store at -20°C
  • TAM Index 11; Store at -20°C
  • TAM Index 12; Store at -20°C
  • TAM Index 13; Store at -20°C
  • TAM Index 14; Store at -20°C
  • TAM Index 15; Store at -20°C
  • TAM Index 16; Store at -20°C
  • Amplification primer mix; Store at -20°C

Reagents for TAM-ChIP anti-mouse conjugate

  • TAM-ChIP anti-mouse antibody conjugate; Store at 4°C
  • pMENTS AM; Store at -20°C
  • Oligonucleotide; Store at -20°C
  • TsTn5 transposase; Store at -20°C
  • 5X Tagmentation Buffer; Store at -20°C
  • TAM Index 1; Store at -20°C
  • TAM Index 2; Store at -20°C
  • TAM Index 3; Store at -20°C
  • TAM Index 4; Store at -20°C
  • TAM Index 5; Store at -20°C
  • TAM Index 6; Store at -20°C
  • TAM Index 7; Store at -20°C
  • TAM Index 8; Store at -20°C
  • TAM Index 9; Store at -20°C
  • TAM Index 10; Store at -20°C
  • TAM Index 11; Store at -20°C
  • TAM Index 12; Store at -20°C
  • TAM Index 13; Store at -20°C
  • TAM Index 14; Store at -20°C
  • TAM Index 15; Store at -20°C
  • TAM Index 16; Store at -20°C
  • Amplification primer mix; Store at -20°C

TAM ChIP Assay Reagents

  • RNase A (10 µg/µl); Store at -20°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • Blocker; 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
  • 10X PBS; Store at -20°C
  • 1 M DTT; Store at -20°C
  • 5X Q5 Reaction Buffer; Store at -20°C
  • Q5 High-Fidelity Polymerase; Store at -20°C
  • dNTPs (10 mM); Store at -20°C
  • Fixation Buffer; Store at 4°C
  • Protein G Agarose beads; Store at 4°C
  • TE, pH 8.0; Store at RT
  • Detergent; Store at RT
  • Stop Solution; Store at RT
  • Chromatin Prep Buffer; Store at RT
  • ChIP Buffer; Store at RT
  • ChIP Buffer without SDS; Store at RT
  • ChIP Filtration Columns; Store at RT
  • Wash Buffer AM1; Store at RT
  • Elution Buffer AM4; Store at RT
  • 10X Elution Buffer AM2; Store at RT
  • 5 M NaCl; Store at RT
  • DNA Purification Elution Buffer; Store at RT
  • Caps and plugs for columns; Store at RT