Fig. 4: CSR-HTGTS reveals that TMEJ repairs unproductive (long-)resected and inversional AID-induced recombination events.

a Schematic representation of activation of CSR in normal B cells stimulated with anti-IgD dextran/LPS/IL-4 which induces AID and activates transcription of Iy1. Deletional CSR events are indicated with a blue arrow and inversional recombination events are indicated with a red arrow. Dotted line indicates intra-switch recombination events. The bait primer used for CSR- HTGTS is situated upstream of Sμ region (b) CSR-HTGTS-seq analysis of break points between 5’Sµ and downstream acceptor S-regions in WT, Polq^−/−, Xrcc4^−/−, Xrcc4^−/− Polq^−/−, Shld1^−/− and Shld1^−/− Polq^−/− splenocytes stimulated with anti-IgD dextran/LPS/IL-4. Junctions obtained via CSR-HTGTS-seq conducted on 3–6 mice, were pooled by genotype and the distribution of total junctions was plotted in 1 kb bins across the Igh. Percentages of mapped deletional and inversional joints located within Sµ and Sy1 are indicated on each distribution plot. To each plot, a zoom into the distribution of AID-junctions situated at Sy1 region, was plotted in 250 bp bins. The total number of plotted junctions (n) is indicated between brackets in the top left corner of each graph. Percentages of mapped deletional and inversional joints within and outside of Sy1 is indicated on each distribution plot. c, d Bars representing mean ± SEM, WT (n = 5), Polq^−/− (n = 6), Xrcc4^−/− (n = 4), Xrcc4^−/− Polq^−/− (n = 3), Shld1^−/− (n = 4) and Shld1^−/− Polq^−/− (n = 3), Unpaired two-tailed T-test, p values are indicated in Source Data file. Percentage of resected Sµ-Sy1 joints in c. Ratios of inversional versus deletional events for each sample in (d). CSR-HTGTS-seq analysis of repair signature of Sµ- Sy1 joints of splenocytes stimulated with anti-IgD dextran/LPS/IL-4. e–g Bars representing mean ± SEM, WT (n = 5), Polq^−/− (n = 6), Xrcc4^−/− (n = 4), Xrcc4^−/− Polq^−/− (n = 3), Shld1^−/− (n = 4) and Shld1^−/− Polq^−/− (n = 3), Unpaired two-tailed T-test, p values are indicated in Source Data file. Percentage of direct joints (e). Percentage of joints containing MH tracks of ≥ 4 bp at break site (f). g MH usage landscapes in each genetic background. Each data point represents the mean ± SEM, WT (n = 5), Polq^−/− (n = 6), Xrcc4^−/− (n = 4), Xrcc4^−/− Polq^−/− (n = 3), Shld1^−/− (n = 4) and Shld1^−/− Polq^−/− (n = 3). Source data are provided as a Source Data file.