Extended Data Fig. 1. Characterization of the mex-6 transcript.

a) IGV genome browser views of sRNAseq reads (wild-type adult hermaphrodites) mapping to the mex-6 locus. rde-11 is a locus that is highly targeted by endogenous sRNAs⁵², shown here for comparison. Genome views are representative of two independent sequencing libraries. b) Maximum projection photomicrographs of pachytene nuclei showing DNA (blue, stained with DAPI), mex-6 RNA (magenta), and puf-5 RNA (green). The mex-6 and puf-5 loci are linked on Chromosome II and, as expected, exhibit closely linked nuclear puncta (arrows). Scale bar is 2.5 µm. Images are representative of 3 worms examined. Results were consistent across four independent FISH experiments. c) Same as in B but showing mex-6 (magenta) and mex-5 (green) RNAs. mex-6 and mex-5 are homologous loci on different chromosomes. Scale bar is 2.5 µm. Images are representative of 3 worms examined. Results were consistent across two independent FISH experiments. The mex-6 and mex-5 puncta do not co-localize, as expected, confirming the specificity of the FISH probes. d) Same as in B and C but using a mex-6 sense probe (magenta) and a puf-5 antisense probe (green). Scale bar is 2.5 µm. Images are representative of 3 worms examined. Results were consistent across two independent FISH experiments. As expected, the mex-6 sense probe does not detect any signal, confirming that the signals detected by the mex-6 antisense probe in panels B and C correspond to RNA and not DNA. Note also the lack of signal under RNAi conditions, suggesting that our FISH protocol does not detect sRNAs.