Extended Data Fig. 7: ACTB and CASP16P antisense sdRNA or DNA sequences do not attract the BRCA1–RNAi and the PALB2–RAD52 repair complexes.

a, RNA–protein binding assay performed in HeLa nuclear extracts using sense or antisense, synthetic biotinylated (or not) ACTB sdRNA (or its DNA sequence) or streptavidin beads only. Representative blot from n = 3 experiments. b, RNA–protein binding assay performed in HeLa nuclear extracts using sense or antisense synthetic biotinylated (or not) CASP16P sdRNA. ACTB sdRNA was used as a positive control. Representative blot from n = 3 experiments. c, RNA–protein binding assay performed in T98G subcellular fractions using synthetic, sense-strand-derived, biotinylated ACTB or CASP16P sdRNA. Representative blot from n = 2 experiments. d, RNA–protein binding assay performed in HeLa nuclear extracts using synthetic biotinylated sdRNAs with the sequence of sense or antisense ACTB sdRNA, ACTB sdRNA (−46) or ACTB sdRNA (+31). See the schematic of the ACTB locus depicting the location of the various sRNAs. ACTB sdRNA (−46) and ACTB sdRNA (+31) are, respectively, located 46 nt upsteam of the 5′ end of the ACTB sdRNA and 31 nt downstream of the 3′ end of the ACTB sdRNA. Representative blot from n = 3 experiments. The beads only were a negative control. All immunoblots were developed using the indicated antibodies. e, f, γ-H2AX ChIP analyses using the data collected from the complementation experiments using sense or antisense ACTB sdRNA, ACTB sdRNA (−46) or ACTB sdRNA (+31) performed in HeLa cells depleted of BRCA1 (e) or DICER1 (f). ChIP results are shown as the mean ± s.e.m. fold change compared to the mock siRNA. n = 2–11 biological replicates (e) and n = 2–7 biological replicates (f). Data were analysed by two-way ANOVA with post hoc Tukey HSD test and compared to the relevant control cells.