Supplementary Figure 1: Localization of DDRNAs at sites of DNA damage is not dependent on H2AX or DDRNA:DNA pairing.

(A) Mapping of the synthetic DDRNA pairs (in blue) L1:L2, U1:U2, U3:U4, T1:T2 relative to the I-SceI cut site. (B–D) Examples of stepwise photobleaching traces of individual DDRNA particles co-localizing with GFP-LacR (relative to Fig. 1B). Raw intensity is depicted in red. Chung-Kennedy non-linear filter is represented in black. (E) Deconvolved DDRNAs display the same localization pattern of the DDRNA pool (relative to Fig. 1B). Plot represents the number of DDRNA molecules at the LacR spot as measured by single-molecule counting based on stepwise photobleaching of fluorescent probes. Dots represent individual cells. The black line represents the mean ± SEM (n = 3 independent experiments). (F,G) Mean fold change of Dicer and Drosha mRNA in NIH2/4 cells by RT-qPCR. Error bars indicate SEM (relative to Fig. 1C, n = 3 independent experiments). (H) DDRNA-Cy5 localization to the damaged site does not depend on H2AX in NIH2/4 cells. The bar plot shows the percentage of cells with DDRNA-TetR co-localization. Error bars indicate SEM (n = 3 independent experiments, ≥80 cells analysed in total per condition). (I) Immunofluorescence analysis (relative to H) confirms reduced γH2AX and 53BP1 foci co-localization with YFP-TetR upon H2AX knockdown in cut NIH2/4 cells (mean of n = 2 independent experiments, ≥40 cells analysed in total per condition). (J,K) DDRNA-Cy5 localization does not depend on DDRNA:DNA pairing. NIH2/4 cells were expressing YFP-TetR and I-SceI and RNase H1-expressing vector or an empty control vector (E.V.) were incubated with DDRNA-Cy5. Cells overexpressing RNase H1 were scored by staining with anti-RNase H1 antibody. In K, the bar plot shows the percentage of cells DDRNA-TetR co-localization. Error bars indicate SEM (n = 3 independent experiments, ≥80 cells analysed in total per condition). (L) Mean fold change of the indicated RNAs in NIH2/4 cells by RT-qPCR. Error bars indicate SEM (relative to Fig. 1D, n = 3 independent experiments). P values were calculated using chi-squared test. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.