Extended Data Fig. 3 |. piRNAi depends on plasmid structure and copy number.

a. Transgenic lines generated by injecting a piRNA cluster either as linear dsDNA (1.5 kb) or the same DNA cloned into a plasmid backbone (high copy ampicillin pTwist vector). The DNA transgenes were generated from cluster_E targeting him-5 and him-8 with six sg-piRNAs. Two-tailed Mann-Whitney tests. *** P = 0.0006, ns = not significant (0.0659). b. him-5 piRNAi plasmid (from panel a) digested with restriction enzymes that cuts in the bacterial vector backbone (left) or undigested (right) was used to induce silencing. The plasmid samples were treated identically (incubated in restriction enzyme buffer with or without restriction enzymes and purified over spin column) and injected at the same concentration. Two-tailed Mann-Whitney test. *** P = < 0.0001. c. Comparison of transgene copy number by whole genome sequencing of transgenic lines with extrachromosomal arrays formed from linear (green) or circular (purple) plasmids. d. Comparison of sg-piRNA expression targeting him-5 from transgenic strains carrying linearized and circular piRNAi transgenes. e. One ‘inactive’ multiplexed piRNAi strain (blue circle in Fig. 3h) was propagated for six generations and scored for males in the population and silencing of germline GFP fluorescence. ‘Generation 0’ corresponds to 2–3 generations after the initial injection. f. Whole-genome sequencing on the strain shown in panel e to determine the copy number of all plasmids in the extra-chromosomal array at the early (generation 1) and late (generation 6) timepoints. Mitochondrial DNA (mtDNA) copy number was used as a control. The copy number was calculated relative to the average sequencing coverage across the entire C. elegans genome. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) him-5: n = 8 (linear), n = 8 (circular), him-8: n = 7 (linear), n = 7 (circular), (b) n = 6 (digested), n = 6 (undigested), (c) n = 3 (all conditions), (d) n = 2 (linear), n = 2 (circular), (e) n = 1, (f) n = 1 biologically independent transgenic strains.