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Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data
Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein–DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that i...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10252487/ https://www.ncbi.nlm.nih.gov/pubmed/37298223 http://dx.doi.org/10.3390/ijms24119271 |
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author | Petrie, Meghan V. He, Yiwei Gan, Yan Ostrow, Andrew Zachary Aparicio, Oscar M. |
author_facet | Petrie, Meghan V. He, Yiwei Gan, Yan Ostrow, Andrew Zachary Aparicio, Oscar M. |
author_sort | Petrie, Meghan V. |
collection | PubMed |
description | Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein–DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a “sensor” for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems. |
format | Online Article Text |
id | pubmed-10252487 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-102524872023-06-10 Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data Petrie, Meghan V. He, Yiwei Gan, Yan Ostrow, Andrew Zachary Aparicio, Oscar M. Int J Mol Sci Article Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein–DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a “sensor” for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems. MDPI 2023-05-25 /pmc/articles/PMC10252487/ /pubmed/37298223 http://dx.doi.org/10.3390/ijms24119271 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Petrie, Meghan V. He, Yiwei Gan, Yan Ostrow, Andrew Zachary Aparicio, Oscar M. Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title | Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title_full | Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title_fullStr | Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title_full_unstemmed | Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title_short | Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data |
title_sort | broadly applicable control approaches improve accuracy of chip-seq data |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10252487/ https://www.ncbi.nlm.nih.gov/pubmed/37298223 http://dx.doi.org/10.3390/ijms24119271 |
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