Protocol for single-cell analysis of DNA double-strand break production and repair in cell-cycle phases by automated high-content microscopy

The mechanisms of DNA double-strand break (DSB) production and repair vary throughout the cell cycle. Here, we provide a protocol to quantify DSB production and repair in G1, S, and G2 phases of asynchronous adherent cells by coupling the staining of DSBs and cell-cycle markers with automated high-content fluorescence microscopy. We describe steps for cell seeding, treatment, staining, imaging, and analysis. This protocol is broadly applicable for monitoring DSB dynamics at single-cell level throughout the cell cycle. For complete details on the use and execution of this protocol, please refer to Geraud et al.¹.