Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

This method, termed Repair Assisted Damage Detection sequencing (RADD-seq), uses repair enzymes to excise the DNA lesions leaving a single-strand gap. The damage sites are then repaired in-vitro with biotinylated nucleotides, followed by DNA fragmentation, immunoprecipitation and sequencing. Reads are mapped back to the reference genome, indicating the locations of damage sites. RADD-seq analysis of damage and repaired samples, as well as an input DNA sample; Final concentration of 50 mM KBrO3 was added to culture medium for one hour. Cells were washed with PBS twice, and genomic DNA was immediately extracted (for damage samples). Alternatively, cell medium was replaced, and cells were allowed to repair for one hour at 37 °C with 5% CO2 before DNA extraction (for repaired samples). Cells were allowed to grow up to a week post KBrO3 treatment, to validate their viability.