Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
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 pa...
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| Format: | Article |
| Language: | English |
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BMC
2017-12-01
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| Series: | BMC Genomics |
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| Online Access: | http://link.springer.com/article/10.1186/s12864-017-4371-5 |
| _version_ | 1811270472050933760 |
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| author | Jian Zhong Zhenqing Ye Samuel W. Lenz Chad R. Clark Adil Bharucha Gianrico Farrugia Keith D. Robertson Zhiguo Zhang Tamas Ordog Jeong-Heon Lee |
| author_facet | Jian Zhong Zhenqing Ye Samuel W. Lenz Chad R. Clark Adil Bharucha Gianrico Farrugia Keith D. Robertson Zhiguo Zhang Tamas Ordog Jeong-Heon Lee |
| author_sort | Jian Zhong |
| collection | DOAJ |
| description | 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 1 ng 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 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. 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. |
| first_indexed | 2024-04-12T22:02:27Z |
| format | Article |
| id | doaj.art-029d36e793094dfa8c6688c1c165354c |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-12T22:02:27Z |
| publishDate | 2017-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-029d36e793094dfa8c6688c1c165354c2022-12-22T03:15:04ZengBMCBMC Genomics1471-21642017-12-0118111010.1186/s12864-017-4371-5Purification of nanogram-range immunoprecipitated DNA in ChIP-seq applicationJian Zhong0Zhenqing Ye1Samuel W. Lenz2Chad R. Clark3Adil Bharucha4Gianrico Farrugia5Keith D. Robertson6Zhiguo Zhang7Tamas Ordog8Jeong-Heon Lee9Epigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo ClinicDivision of Biomedical Statistics and Informatics, Department of Health Science Research, Mayo ClinicEpigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo ClinicEpigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo ClinicDivision of Gastroenterology and Hepatology, Department of Medicine, Mayo ClinicEnteric Neuroscience Program, Mayo ClinicDepartment of Molecular Pharmacology and Experimental Therapeutics, Mayo ClinicDepartment of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia UniversityDivision of Gastroenterology and Hepatology, Department of Medicine, Mayo ClinicEpigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo ClinicAbstract 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 1 ng 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 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. 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.http://link.springer.com/article/10.1186/s12864-017-4371-5Nanogram DNAChIP-seqDNA purificationDNA storage |
| spellingShingle | Jian Zhong Zhenqing Ye Samuel W. Lenz Chad R. Clark Adil Bharucha Gianrico Farrugia Keith D. Robertson Zhiguo Zhang Tamas Ordog Jeong-Heon Lee Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application BMC Genomics Nanogram DNA ChIP-seq DNA purification DNA storage |
| title | Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application |
| title_full | Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application |
| title_fullStr | Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application |
| title_full_unstemmed | Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application |
| title_short | Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application |
| title_sort | purification of nanogram range immunoprecipitated dna in chip seq application |
| topic | Nanogram DNA ChIP-seq DNA purification DNA storage |
| url | http://link.springer.com/article/10.1186/s12864-017-4371-5 |
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