Precise Mapping of Physiological DSBs Using In-Suspension Break Labeling In Situ and Sequencing (sBLISS)

DNA double-strand breaks (DSBs) are a major source of genomic instability. Physiological DSBs are naturally occurring breaks that happen during normal cellular processes. Unlike DNA breaks resulting from DNA damage due to external factors like radiation or chemicals, physiological DSBs play critical roles in various normal biological functions. Some key processes involving physiological DSBs include V(D)J recombination, transcription, and replication. These breaks are typically tightly controlled and are part of the cellular machinery designed to maintain and enhance genomic integrity and diversity. However, if these breaks are misrepaired or left unrepaired, they can contribute to genomic instability, potentially leading to senescence and diseases such as cancer. Here, we outline various methods commonly employed to detect physiological DSBs and introduce a detailed, step-by-step protocol for mapping these breaks using the in-suspension break labeling in situ and sequencing (sBLISS) technique. sBLISS offers single nucleotide resolution and is versatile enough to be applied to any cell type amenable to single-cell suspension. This comprehensive approach not only enhances our understanding of DSBs but also aids in the exploration of their roles in genomic instability.