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Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Published on: June 3, 2017
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An optimized chromatin immunoprecipitation protocol using Staph-seq for analyzing genome-wide protein-DNA
Fang Tao1, Egidy Rhonda2, Xi He2
1Children's Research Institute, Children's Mercy Kansas City, Kansas City, MO 64108, USA; Stowers Institute for Medical Research, Kansas City, MO 64111, USA.
STAR Protocols
|January 3, 2023
Summary
This study introduces an optimized protocol for chromatin immunoprecipitation sequencing (ChIP-seq) to improve the detection of low-abundant transcription factors (TFs) bound to the genome. The Staph-seq method enhances sensitivity and reduces contamination for accurate TF mapping.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Understanding transcription factor (TF) functions relies on genome-wide mapping.
- Low-abundant TFs transiently binding the genome pose a challenge for standard ChIP-seq assays.
Purpose of the Study:
- To present an optimized protocol for efficient chromatin complex pull-down.
- To enhance the sensitivity of genome-wide TF binding detection.
Main Methods:
- Development of an optimized chromatin immunoprecipitation sequencing (ChIP-seq) protocol using Staph-seq.
- Implementation of double size selection for improved DNA-protein complex capture.
- Mitigation of Staphylococcal contamination common in other ChIP-seq protocols.
Main Results:
- The Staph-seq protocol efficiently captures genome-wide protein-DNA associations.
- The method demonstrates enhanced sensitivity for detecting low-abundant TF binding.
- Potential Staphylococcal contamination is effectively eliminated.
Conclusions:
- The optimized Staph-seq protocol provides a sensitive and reliable method for genome-wide TF mapping.
- This technique facilitates the study of transiently bound, low-abundant transcription factors.
- The protocol addresses common contamination issues in ChIP-seq assays.
