N2-alkyl-dG lesions trigger R-loop formation in genome

Strand-specific kethoxal-assisted single-stranded DNA (ssDNA) sequencing (spKAS-seq) for mapping R-loop formation in HEK293T cells with/without the N2-heptynyl-dG treatment . Overall design: spKAS-seq was conducted following previously published procedures (PMID: 36449625) with minor modifications, where N2-heptynyl-dG was added to the culture medium until its final concentration reached 10 µM. After a 3-h incubation, the cells were treated with 5 mM N3-kethoxal dissolved in the culture medium for 10 min at 37°C, then harvested the genomic DNA for mapping the R-loop formation.