Journal: Nature Communications
Article Title: qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing
Figure Lengend Snippet: Quantitative DSB sequencing (qDSB-Seq) method. a A comparison of current DNA double-strand break (DSB) counting (e.g., immunofluorescence microscopy, quantitative PCR (qPCR)) and DSB sequencing strategies (e.g., BLESS 3 , i-BLESS 15 , END-Seq 6 , Break-Seq 4 , DSBCapture 5 ) with our qDSB-Seq method. b In qDSB-Seq protocol after DSB induction cells are treated with a restriction enzyme to introduce site-specific, infrequent DSBs (spike-ins). Next, DSBs are labeled (here using i-BLESS 15 or BLESS 3 ) and sequenced. Simultaneously, genomic DNA (gDNA) sequencing (or qPCR) is performed and used to estimate the cutting efficiency of the enzyme, and thus frequency of induced spike-in DSBs, which is then used to quantify the absolute DSB frequencies (per cell) of studied DSBs in the sample (Methods). c qDSB-Seq can be combined with any sequencing-based DSB labeling method. d , e Spike-in DSBs were induced in two different ways: d the studied cells were digested using the NotI, SrfI, AsiSI, or BamHI restriction enzyme in vitro; e cells expressing a restriction enzyme in vivo were mixed with the studied cells (I-SceI digestion) or alternatively a restriction enzyme was expressed in vivo in all studied cells (DIvA cells discussed below)
Article Snippet: Samples were treated with NotI (NEB, Thermo Scientific), SrfI (NEB), AsiSI (NEB), or BamHI (Thermo Scientific) for 1 h at 37 °C.
Techniques: Sequencing, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Introduce, Labeling, In Vitro, Expressing, In Vivo