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Updated: Apr 15, 2026
Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq
Published on: April 19, 2013
ChIP on chip and ChIP-Seq assays: genome-wide analysis of transcription factor binding and histone modifications
Smitha Pillai1, Srikumar P Chellappan
1Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL, 33612, USA.
Abstract:
Deregulation of transcriptional activity of many genes has been causatively linked to human diseases including cancer. Altered patterns of gene expression in normal and cancer cells are the result of inappropriate expression of transcription factors and chromatin modifying proteins. Chromatin immunoprecipitation assay is a well-established tool for investigating the interactions between regulatory proteins and DNA at distinct stages of gene activation. ChIP coupled with DNA microarrays, known as ChIP on chip, or sequencing of DNA associated with the factors (ChIP-Seq) allow us to determine the entire spectrum of in vivo DNA binding sites for a given protein. This has been of immense value because ChIP on chip assays and ChIP-Seq experiments can provide a snapshot of the transcriptional regulatory mechanisms on a genome-wide scale. This chapter outlines the general strategies used to carry out ChIP-chip assays to study the differential recruitment of regulatory molecules based on the studies conducted in our lab as well as other published protocols; these can be easily modified to a ChIP-Seq analysis.
Insights
Chromatin immunoprecipitation (ChIP) assays reveal gene regulatory mechanisms. ChIP coupled with DNA microarrays (ChIP on chip) or sequencing (ChIP-Seq) map protein-DNA interactions genome-wide.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Gene expression deregulation is linked to human diseases like cancer.
- Altered gene expression patterns result from aberrant transcription factors and chromatin modifiers.
- Understanding these regulatory proteins is crucial for disease research.
Purpose of the Study:
- To outline strategies for Chromatin Immunoprecipitation (ChIP) assays.
- To detail methods for studying regulatory molecule recruitment.
- To adapt protocols for both ChIP on chip and ChIP-Sequencing (ChIP-Seq) analyses.
Main Methods:
- Chromatin immunoprecipitation (ChIP) assay to investigate protein-DNA interactions.
- ChIP coupled with DNA microarrays (ChIP on chip) for genome-wide binding site determination.
- ChIP coupled with DNA sequencing (ChIP-Seq) for comprehensive mapping of protein-DNA interactions.
Main Results:
- ChIP assays are effective for studying regulatory protein-DNA interactions.
- ChIP on chip and ChIP-Seq provide genome-wide insights into transcriptional regulation.
- These methods offer a snapshot of regulatory mechanisms on a large scale.
Conclusions:
- ChIP-based techniques are invaluable for dissecting gene regulation.
- The outlined strategies facilitate the study of differential recruitment of regulatory molecules.
- Protocols are adaptable for both ChIP on chip and ChIP-Seq applications.
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