Fig. 1

i-BLESS method and its validation. a i-BLESS workflow. Briefly, cells are encapsulated in agarose beads, lysed and deproteinated, DSBs are labeled with a biotinylated adapter (proximal) and captured on streptavidin. Free ends of DNA fragments are ligated to the second adapter (distal), and the resulting fragments are amplified and sequenced. b Impact of experimental protocol parameters on quality of i-BLESS data. mec1-1 sml1-1 cells were treated with hydroxyurea and subjected to indicated treatments: intensive fixation: cell fixation with 2% formaldehyde for 30 min; gentle fixation: cell fixation with 2% formaldehyde for 5 min; storage: storage of fixed cells for 7 days at 4 °C; intensive proteinase K: 50 µg mL⁻¹ overnight at 50 °C; and gentle proteinase K: 1 µg mL⁻¹ for 5 min at 37 °C. For each sample, i-BLESS signal around replication origins (dotted vertical lines) in a representative region of chromosome VII, autocorrelation of i-BLESS signal, cross-correlation of i-BLESS data with MNase-seq data¹⁸ and averaged i-BLESS signal around replication origins are shown. i-BLESS data in the top two panels, for which signal-to-noise ratio is the lowest (as illustrated by averaged meta-profiles of i-BLESS signal around replication origins), shows clear periodicity in autocorrelation pattern related to nucleosome spacing, suggesting over-fixation as a main source of noise during DSB detection. Reads were normalized to 1 million total reads. c Cross-correlation of i-BLESS data with nucleosome positioning data (MNase-seq) characteristic for DSBs located preferentially between nucleosomes (left) or within nucleosomes (right). As MNase signal is increased in nucleosome depleted regions, a peak for cross-correlation observed at position 0 bp (left panel) implies DSBs enriched between nucleosomes, while peaks observed at positions +/−80 bp (right panel) indicate DSBs enriched within nucleosomes. d Averaged i-BLESS signal in a 22 bp window around BamHI cutting sites (marked with red arrows). e Number of i-BLESS reads at NotI (5′ overhangs), SrfI (blunt ends) and AsiSI (3′ overhangs) recognition sites in wild type cells treated with all 3 enzymes simultaneously. Median (center line), lower/upper quartiles (box limits), and lower/upper adjacent (whiskers) are shown