Preparation and Analysis of GLOE-Seq Libraries for Genome-Wide Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions

GLOE-Seq is a next-generation sequencing method for the genome-wide mapping of 3′-OH termini, either resulting from single- or double-strand breaks or introduced by enzymatic conversion of lesions or modified nucleotides. This protocol provides instructions for isolation of genomic DNA from budding yeast or mammalian cells, preparation of libraries for sequencing, and data analysis by the associated computational pipeline, GLOE-Pipe. It is optimized for the Illumina next-generation sequencing platform and can be adapted to intact genomic DNA of any origin. For complete details on the use and execution of this protocol, please refer to Sriramachandran et al. (2020).