GSE134219
Genomewide nucleotide-resolution mapping of single-strand breaks and lesions by GLOE-Seq [digestion]
Numerous methods are available for the mapping of DNA lesions ranging from double-strand breaks (DSBs) to incorporated ribonucleotides. We now present a technology based on the Genomewide Ligation of 3'-OH Ends (GLOE-Seq) and an associated computational pipeline designed for single-stranded breaks (SSBs), but versatile enough to be applied to any lesion that is convertible into a free 3'-OH terminus. We demonstrate its applicability to the mapping of Okazaki fragments without prior size selection and detect biases and asymmetries in the distribution of spontaneous SSBs in budding yeast and human DNA. Overall design: I. Method validation with yeast genomic DNA   GLOE-Seq was validated by mapping DNA single- and double-strand breaks generated by restriction or nicking endonucleases in yeast genomic DNA. The sensitivity of the method was assessed by dilution of nicked with untreated DNA at defined ratios. Benchmarking against other methods was performed by direct comparison to samples prepared by following the EndoSeq protocol. The genomewide distribution of single-strand breaks was analysed in samples of undigested genomic DNA, using control samples of randomly fragmented DNA for comparison. Two replicates were used per conditions, except for Nb.BsrDI-treated DNA (4 replicates) and EndoSeq samples (1 replicate).