File:BLESS workflow (update v2).png

English: Double-stranded DNA breaks (DSBs) are labeled in situ with proximal DNA hairpin linkers containing a biotin marker. Cells are fixed, lysed and treated with proteinases for extraction and subsequent shearing of genomic DNA (gDNA). Labeled and unlabeled gDNA fragments are passed through streptavidin-derived beads, which capture the labeled fragments with high specificity due to the strong affinity of biotin markers to streptavidin. After passage through streptavidin beads, unlabeled gDNA fragments are removed, leaving the enriched biotin-labeled gDNA fragments. Distal linker is ligated to labeled gDNA fragments at the free end. I-SceI endonuclease cuts linkers at the restriction site to release gDNA fragments from biotin. Barcode-specific primers are used for amplification of enriched fragments by Polymerase Chain Reaction (PCR). Next-Generation Sequencing of PCR products is then used for single nucleotide-resolution analysis of DSBs in the genome.