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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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High-resolution, strand-specific R-loop mapping via S9.6-based DNA-RNA immunoprecipitation and high-throughput
Lionel A Sanz1, Frédéric Chédin2
1Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA, USA. flchedin@ucdavis.edu.
Nature Protocols
|May 5, 2019
Summary
Researchers developed two methods, DRIP-seq and DRIPc-seq, to map R-loops, which are DNA-RNA structures linked to genomic instability and diseases like cancer. DRIPc-seq offers high-resolution, strand-specific mapping.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- R-loops are three-stranded DNA structures (RNA-DNA hybrid and single DNA strand) crucial for nuclear processes.
- R-loop dysregulation is associated with genomic instability and human diseases, including cancer.
- Accurate mapping of R-loops is essential for understanding their biological roles and pathological implications.
Purpose of the Study:
- To describe two immunoprecipitation-based methods for mapping R-loop structures.
- To introduce DRIPc-seq as a high-resolution, strand-specific alternative to basic DRIP-seq.
Main Methods:
- Developed DRIP-seq (DNA-RNA immunoprecipitation followed by sequencing) for R-loop mapping.
- Developed DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion and sequencing) for high-resolution, strand-specific mapping.
- Both methods utilize the S9.6 antibody for immunoprecipitation of RNA-DNA hybrids after DNA extraction and digestion.
Main Results:
- DRIP-seq is a robust but resolution-limited method for R-loop mapping.
- DRIPc-seq provides high-resolution, strand-specific R-loop mapping by sequencing the RNA moiety.
- DRIPc-seq is a rapid (5-day) method applicable to various cell types.
Conclusions:
- DRIPc-seq enables accurate, genome-wide R-loop distribution mapping.
- Understanding R-loop dynamics through precise mapping is vital for elucidating their formation, functions, and resolution.
- These methods are crucial tools for investigating the role of R-loops in nuclear processes and disease.
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