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

BMC Genomics

Methodology

Keith D. Robertson¹  Zhiguo Zhang²  Zhenqing Ye³  Adil Bharucha⁴  Tamas Ordog⁵  Gianrico Farrugia⁶  Jian Zhong⁷  Samuel W. Lenz⁷  Chad R. Clark⁷  Jeong-Heon Lee⁸ 

[1] Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 55905, Rochester, MN, USA;Department of Biochemistry and Molecular Biology, Mayo Clinic, 55905, Rochester, MN, USA;Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, 10032, New York, NY, USA;Division of Biomedical Statistics and Informatics, Department of Health Science Research, Mayo Clinic, 55905, Rochester, MN, USA;Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Enteric Neuroscience Program, Mayo Clinic, 55905, Rochester, MN, USA;Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Department of Physiology and Biomedical Engineering, Mayo Clinic, 55905, Rochester, MN, USA;Enteric Neuroscience Program, Mayo Clinic, 55905, Rochester, MN, USA;Epigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Epigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, 55905, Rochester, MN, USA;Department of Biochemistry and Molecular Biology, Mayo Clinic, 55905, Rochester, MN, USA;Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, 55905, Rochester, MN, USA;

关键词: Nanogram DNA;    ChIP-seq;    DNA purification;    DNA storage;   

DOI  :  10.1186/s12864-017-4371-5

 received in 2017-08-29, accepted in 2017-12-08,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundChromatin 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 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored.ResultsWe compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng 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 1 ng 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© The Author(s). 2017