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Tech Note

Library generation for ChIP-seq analysis of transcription factors with the ThruPLEX DNA-seq kit

Introduction Results Conclusion Methods References

Introduction  

Chromatin immunoprecipitation sequencing (ChIP-seq) is an invaluable tool for interrogating genomic loci that are targeted by DNA-binding proteins of interest. One particularly useful application is identifying the binding sites of transcription factors, which are responsible for the determination of cell fate and play a key role in development. However, as transcription factors involved in cell fate may only be expressed in specific or rare cell type(s), ChIP-seq experiments can be challenging due to highly limited samples.

To address this difficulty, Dr. Yuri Yasuoka (RIKEN Biomedical Science Research Center) used our ThruPLEX DNA-Seq Kit to generate sequencing libraries for efficient ChIP-seq analysis of transcription factors from ~250 Xenopus gastrulae.

Results  

The researchers generated ThruPLEX sequencing libraries from ~250 Xenopus gastrulae using either total ("Input") or Otx2-immunoprecipitated (anti-Ox2; "Otx2 ChIP") DNA. ThruPLEX libraries were sequenced on an Illumina NextSeq® 550 instrument with 75-bp paired-end reads and mapped to the Xenopus genome using the Burrows-Wheeler aligner. Sequencing metrics demonstrated comparable total reads, mapped reads, and mapped read percentages between Input and Otx2 ChIP samples (Table I).

SampleTotal readsMapped reads (%)
Input 74,426,066 70,219,181 (94.35%) 
Otx2 ChIP 82,794,598 75,710,767 (91.44%)

Table I. Sequencing metrics of ThruPLEX DNA-seq libraries derived from total (Input) and immunoprecipitated (Otx2 ChIP) chromatin samples.

Mapped reads from Otx2 ChIP samples corresponding to regions of interest were visualized with the Integrative Genomics Viewer (IGV) (Figure 1). Sequencing mapping results were concordant with a prior ChIP-seq study (Yasuoka et al. 2014). Notably, this prior study used a number of Xenopus gastrulae (~2,000–3,000) that is an order of magnitude greater than the present study (~250 gastrulae). The researchers also noted that a sufficient amount of library was able to be generated from ~1 ng of DNA (12 PCR cycles in the ThruPLEX protocol), speaking to the exquisite sensitivity of our ThruPLEX DNA-Seq Kit.

ChIP-seq data derived from Xenopus gastrulae using our ThruPLEX DNA-Seq kit

Figure 1. Visualization of Otx2 ChIP samples using IGV.

Conclusion  

While ChIP-seq is an extremely powerful tool for querying the binding domain(s) of DNA-binding proteins, sample input requirements can present significant hurdles, particularly for rare targets. In the present study, our ThruPLEX chemistry was used to generate ChIP-seq libraries from ~250 Xenopus gastrulae, representing a ~10-fold improvement over a prior study that did not use our ThruPLEX chemistry (Yasuoka et al. 2014). Based on yield and sensitivity, the researchers noted that, depending on the specific antibody and its target, it may be feasible to use inputs of as little as 50 gastrulae. These results demonstrate the ability of our ThruPLEX DNA-Seq Kit to generate DNA-seq libraries from extremely limited samples and present a highly sensitive method of querying rare targets in ChIP-seq.

Methods  

Chromatin extraction and shearing

Approximately 250 Xenopus gastrulae were fixed with 1% formalin and stored at –80°C until processing. Fixed gastrulae were then lysed with lysis buffer, nuclei were concentrated, and chromatin was fragmented with a Picorupter sonication device (Diagenode). A fraction of fragmented chromatin was saved for the Input sample.

Chromatin immunoprecipitation and DNA purification

An anti-Otx2 antibody was conjugated to Protein A magnetic beads, antibody-bound beads were added to the fragmented chromatin, and the mixture was allowed to incubate overnight at 4°C with rotation. Beads were then washed once with RIPA buffer and incubated at 65°C overnight in elution buffer containing 1% SDS. Following RNase A treatment and decrosslinking with Proteinase K, DNA was purified with a MinElute PCR Purification Kit (QIAGEN) using two elutions of 10 µl each. Recovered DNA was quantified using a dsDNA HS kit (Qubit).

Library preparation and sequencing

DNA-seq libraries were prepared with the ThruPLEX DNA-Seq Kit using 10 µl of either Otx2 ChIP or Input DNA. ThruPLEX libraries were sequenced on an Illumina NextSeq 550 with 75-bp paired-end reads.

Data analysis

Sequencing reads were mapped to the Xenopus genome (v9) using the Burrows-Wheeler aligner, and genomic regions were visualized with the Integrative Genomics Viewer (Broad Institute).

References  

Yasuoka, Y. et al. Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification. Nat. Commun. 5, 4322 (2014).

Related Products

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R400674 ThruPLEX® DNA-Seq Kit 24 Rxns USD $705.00

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ID Number  
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 24 reactions.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400674: ThruPLEX DNA-Seq Kit

R400674: ThruPLEX DNA-Seq Kit
R400675 ThruPLEX® DNA-Seq Kit 48 Rxns USD $1360.00

License Statement

ID Number  
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 48 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400675: ThruPLEX DNA-Seq Kit

R400675: ThruPLEX DNA-Seq Kit
R400676 ThruPLEX® DNA-Seq Kit 96 Rxns USD $2493.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 96 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400676: ThruPLEX DNA-Seq Kit

R400676: ThruPLEX DNA-Seq Kit
R400677 ThruPLEX® DNA-Seq Kit 480 Rxns USD $11040.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product is composed of five 96-reaction kits (Cat. No. R400676), 480 reactions total, of ThruPLEX DNA-Seq reagents.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400677: SMARTer ThruPLEX DNA-Seq Kit

R400677: SMARTer ThruPLEX DNA-Seq Kit

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