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. Author manuscript; available in PMC: 2013 Nov 10.
Published in final edited form as: Cell. 2012 Nov 9;151(4):885–899. doi: 10.1016/j.cell.2012.10.022

Figure 4.

Figure 4

Assays for siRNAs emanating from direct and indirect targets of an actin 3' UTR dsRNA trigger. (A) Experimental design. RNAi against a member of a partially-redundant multigene family is induced using a dsRNA trigger uniquely matching the 3' UTR of one of the gene family. For regions of mismatch between the highly similar coding regions, siRNA populations that derive from the direct target gene (act-1 in this case) can be distinguished by sequence from potential tertiary siRNA populations that derive from the remaining genes in the family. (B–D) Genome-wide gene by gene comparison of antisense siRNA matches. Each point represents one C. elegans mRNA transcript from a duplicate-subtracted reference sequence set (Wormbase). Vertical position on each plot represents antisense siRNA counts for that gene in small RNA captured following act-1 3' UTR RNAi as shown in A. Horizontal positions represent antisense siRNA counts from comparable RNAi experiments with non-actin triggers. (B) Horizontal axis: sel-1 mismatched trigger (see Figure 1), (C, D) Horizontal axis: ben-1 triggers R1934U and R1934D (Figure 3). (E) Distribution of antisense siRNAs matching the act-1 transcript following RNAi feeding with the act-1 3' UTR trigger (blue line). Coverage at each base position is normalized to aggregate antisense siRNA counts for all C. elegans genes, and a smoothing window of 61b is used. Enrichment of antisense counts throughout the act-1 gene is evident in comparison to control samples in which RNAi was triggered to a non-actin target (green, red, orange lines). (F) Equivalent composite coverage plot of antisense siRNAs deriving from act-2, act-3, act-4, or act-5 and not from act-1. Given the high signal of act-1 matching siRNAs in E and the known error frequency in high throughput RNA-seq methods, there is some possibility that a fraction of the signal in F represents mis-sequencing of act-1-matched siRNAs. This value is estimated (magenta line, F) by generating a series of 72 "faux-actin" genes (Supplementary Experimental Procedures). (See also Figure S3.)