Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing

Area of Science:

• Genomics
• Molecular Biology
• DNA Repair

Background:

• DNA double-strand breaks (DSBs) are critical lesions.
• Understanding DSB repair mechanisms is vital for genome stability.
• Genome-wide identification of DSBs is challenging.

Purpose of the Study:

• To present a protocol for mapping genome-wide DSB sites.
• To utilize translocation capture sequencing for DSB site identification.
• To assess DNA repair mechanisms and accessibility of DSBs.

Main Methods:

• Introduction of bait DSBs using Cas9 nuclease.
• Repair of bait DSBs by the host cell, linking to other DSBs.
• Isolation of bait site DNA, enrichment of off-site repair, and next-generation sequencing.

Main Results:

• A protocol for translocation capture sequencing is detailed.
• The method enables mapping of DSB sites across the genome.
• Facilitates identification of DSBs accessible to DNA repair machinery.

Conclusions:

• Translocation capture sequencing is a powerful tool for DSB analysis.
• The protocol aids in studying DNA repair pathways.
• Enables genome-wide identification of double-strand break locations.