Extended Data Fig. 1. Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation.

a, RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b, Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c, Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d, Quantification of fluorescence signal from HeLa cells treated as in c. Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e, Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly ³²P-radiolabelled ssRNA and dsRNA substrates. Signals were visualized and quantified by PhosphorImager. The level of immunoprecipitation signal is shown and expressed as the percentage of input. Images and data are representative of two experiments. For gel source data, see Supplementary Fig. 1. f, Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g, Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.