Protocol for mapping sister chromatid exchanges and structural variations using sci-L3-strand-seq

Understanding spontaneous genome rearrangement requires mapping both error-free and mutational repair outcomes, particularly error-free sister chromatid exchanges (SCEs). Here, we present a sci-L3-Strand-seq protocol capturing SCEs across thousands of single cells from 12–16 independent samples in one experiment. We detail steps for cell labeling, sample banking, nucleosome depletion, in situ combinatorial barcoding, enrichment of BrdU-labeled cells, and nascent strand ablation. We then detail linear amplification, reverse transcription, and second-strand synthesis. Libraries are typically sequencing-ready within three days from frozen cells. For complete details on the use and execution of this protocol, please refer to Chovanec et al. 1