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Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing
Osama Hidmi1, Sara Oster1, Diala Shatleh1
1The Concern Foundation Laboratories, The Lautenberg Center for Immunology and Cancer Research, Department of Immunology and Cancer Research-IMRIC, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.
STAR Protocols
|May 8, 2024
Summary
This study introduces a new method, single-nucleotide resolution in-suspension break labeling in situ and sequencing (sBLISS), to map physiological double-stranded breaks (DSBs). This technique aids in understanding genomic instability and DSB formation.
Area of Science:
- Genomics
- Molecular Biology
- Genomic Instability
Background:
- Physiological double-stranded breaks (DSBs) are critical drivers of genomic instability.
- Understanding the precise locations and formation of DSBs is essential for comprehending genome maintenance and disease pathogenesis.
Purpose of the Study:
- To present a novel protocol for high-resolution mapping of physiological DSBs.
- To enable the study of factors influencing DSB formation and their genomic distribution.
Main Methods:
- Development and description of the in-suspension break labeling in situ and sequencing (sBLISS) protocol.
- Detailed steps include cell fixation, DSB labeling, DNA isolation, in vitro transcription (IVT), reverse transcription, and library preparation.
- Achieves single-nucleotide resolution for DSB mapping.
Main Results:
- The sBLISS protocol provides a comprehensive map of DSBs across the genome.
- Demonstrates the utility of sBLISS for investigating the role of various factors in DSB generation.
- Offers a powerful tool for studying the landscape of DNA damage and repair.
Conclusions:
- sBLISS is an effective method for mapping physiological DSBs at single-nucleotide resolution.
- This protocol facilitates research into the mechanisms underlying genomic instability.
- Provides a foundation for future studies on DNA damage response pathways.
