Igk locus capture-based target enrichment sequencing

Cell cycle is a major determinant of DNA double-strand break (DSB) repair pathway choice with homologous recombination (HR) that is most active in S phase cells and non-homologous end-joining (NHEJ) that dominates in G1 phase cells. A third less well-defined mechanism, 'alternative end-joining', has been shown to promote error-prone repair in NHEJ- or HR-deficient cells. Here, we have used a physiologic system of NHEJ-mediated genomic rearrangements induced by the site-specific RAG1/2 endonuclease in G1 cells to investigate the fate of unrepaired G1 DSBs upon entry into the cell cycle. We show that, in the absence of XRCC4, alternative end-joining rescues RAG-induced DSB repair and promotes chromosome translocations upon G1 cell cycle exit. Overall design: Coding and signal sequences of 140 mouse Igk V and 5 Igk J segments were downloaded from MGI database. 280 bps windows flanking recombination signal site (RSSs) of Igk V and J segments, including coding and signal sequences, were used to design Agilent SureSelect customized probes (Agilent Technologies). A total of 2348 120-mer probes (1168 for signal ends; 1180 for coding ends) were designed for Igk locus capture. SureSelectXT Target Enrichment System kit (Agilent Technologies) was used to prepare Igk locus capture sequencing libraries.