Supplementary Figure 8: Mime-seq can be performed in wild-type background in worms and flies.

(a) Expression of Ath-HEN1 in the ASE neurons was monitored by joint expression of mCherry-H2B using a T2A peptide. At least 10 animals were analyzed from each of two independent transgenic lines. Scale bar = 10 μm. (b) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in henn-1(0) worm L1s expressing Ath-HEN1 under the ASE-specific promoter, before and after oxidation treatment. Reproducibility of the two biological replicates, for both treated and untreated conditions, are shown. Spearman correlation coefficients and their significance are shown. (c) Same as in (b) but with animals carrying wild-type (wt) endogenous henn-1. Experiments in this figure were performed on two biologically independent samples. (d) Venn diagram shows the overlap between the significantly enriched miRNAs (log₂ Fold Change>1, p-Value<0.001 for henn-1(tm4477) or <0.01 for wt) in each of the two backgrounds. (e) Muscle mime-seq results from C. elegans from both henn-1(0) and wild-type backgrounds are shown as volcano plots. These show miRNAs enriched or depleted following oxidation treatment, and their associated p-Values. The wt enrichments shown are after subtraction of the background enrichment in non-transgenic, wt animals to account for endogenous Cel-HENN-1-mediated methylation. The Venn diagram shows the overlap between the significantly enriched miRNAs (log₂ fold-change>1, p-Value<0.001 for henn-1(0) or <0.01 for wt) in each of the two backgrounds. Experiments in this figure were performed on two biologically independent samples. (f) Muscle mime-seq results from Drosophila using all muscle or flight-muscle-specific drivers are shown as volcano plots. These show miRNAs enriched or depleted following oxidation treatment, and their associated p-Values. The Venn diagram shows the overlap between the miRNAs enriched >2-fold in each of the two experiments. Experiments in this figure were performed in single replicates.