Protocol for cyclic DNA-damaging treatments to generate high mutational burden in the human A549 cell line

Here, we present a protocol that describes iterative exposure of the human lung cancer cell line A549 to DNA-damaging agents to progressively increase mutational burden. We describe steps for controlled cytotoxic dosing, metabolic activation, and repeated treatment cycles of up to 70 iterations, followed by clonal isolation. This approach enables the generation of highly mutated cell populations suitable for downstream genomic analyses. For complete details on the use and execution of this protocol, please refer to Juhász et al.¹.