Biotinylated proximal linker

After 4OHT and or auxin treatment in G1 synchronized cells, DSBs labelling was carried out using BLESS method as described previously37 (link). Briefly, cells were fixed with formaldehyde, lysed and mildly digested with proteinase K at 37ºC in order to purify intact nuclei, then DSBs were blunted and 5'phosphorylated using Quick Blunting Kit (NEB). Subsequently, a biotinylated proximal linker (Sigma) was ligated to DSBs using T4 ligase (NEB). Next, DNA was extracted by precipitation with isopropanol and sonicated using Covaris S220 ultrasonicator to create fragments approximately 400bp long. Labeled, biotinylated fragments were captured on streptavidin beads (Invitrogen) and ligated to a distal linker (Sigma). Resulting DNA fragments were then linearized by I-SceI (NEB) digestion and amplified by PCR. Libraries were prepared using TruSeq DNA LT Sample Prep Kit (Illumina), followed by 2x61 bp and 2x70 bp next generation sequencing on Illumina HiSeq 2500. Raw data were filtered using BLESS adapter in order to keep only the end proximal to the cut from the paired read. These proximal reads were aligned using bwa-aln and PCR-duplicates were cleaned using samtools. The coverage and the normalization by the total number of reads was then generated using R. For each site we computed the average level of normalized counts in a window of +/-500bp around sites.