Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

Jian Zhong, Zhenqing Ye, Samuel W. Lenz, Chad R. Clark, Adil Eddie Bharucha, Gianrico Farrugia, Keith D Robertson, Zhiguo Zhang, Tamas Ordog, Jeong Heon Lee

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Background: Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. Results: We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. Conclusions: This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Original languageEnglish (US)
Article number985
JournalBMC Genomics
Volume18
Issue number1
DOIs
StatePublished - Dec 21 2017

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Chromatin Immunoprecipitation
DNA
Gene Library
Libraries
Buffers
Polypropylenes
Chloroform

Keywords

  • ChIP-seq
  • DNA purification
  • DNA storage
  • Nanogram DNA

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application. / Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W.; Clark, Chad R.; Bharucha, Adil Eddie; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong Heon.

In: BMC Genomics, Vol. 18, No. 1, 985, 21.12.2017.

Research output: Contribution to journalArticle

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AU - Bharucha, Adil Eddie

AU - Farrugia, Gianrico

AU - Robertson, Keith D

AU - Zhang, Zhiguo

AU - Ordog, Tamas

AU - Lee, Jeong Heon

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AB - Background: Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. Results: We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. Conclusions: This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

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KW - DNA storage

KW - Nanogram DNA

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