C. elegans husbandry
All C. elegans strains (Supplementary Table 1) were maintained at 20 °C on nematode growth medium (NGM) plates seeded with E. coli OP50 according to previously described methods51, unless otherwise specified. Strains obtained from the Caenorhabditis Genetics Center are listed in Supplementary Table 1. WT refers to the N2 Bristol strain. All strains are available upon reasonable request. All experiments were performed with age-matched C. elegans hermaphrodites. Sample sizes were not predetermined by statistical methods. Randomization was not applied, as experiments were designed according to genotype or condition. Investigators were not blinded to allocation, but control and experimental samples were processed in parallel under identical conditions. No ethical approval is required to work with C. elegans.
Cell lines
HEK293T cells (CRL-3216; sourced and authenticated by the American Type Culture Collection) and HEK293T-derived reporter and knockout cell lines (Supplementary Table 1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-065) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10437-028). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO 2 . All cells used in this study were tested negative for mycoplasma contamination, and no commonly misidentified cell lines were used.
RNAi
Feeding RNAi
Gravid adults were treated with alkaline-bleach solution (20% commercial bleach, 0.5 M NaOH) to isolate embryos (egg preparation). The embryos were then transferred to RNAi plates (NGM plates containing 1 mM IPTG and 25 mg ml–1 carbenicillin) seeded with E. coli HT115 expressing either control dsRNA (L4440 empty vector) or dsRNAs targeting specific genes (utp-20 or dpy-6). The utp-20 and dpy-6 RNAi colonies are from the Ahringer library and all plasmid identities were confirmed by Sanger sequencing and whole plasmid sequencing (Supplementary Table 1).
esiRNA treatment
Dried esiRNA oligonucleotides targeting RTCB (Sigma Aldrich, EHU009301-20UG) were resuspended in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). esiRNA targeting RLUC (Sigma Aldrich, EHURLUC-50UG) was used as a negative control in all RNAi experiments. HEK293T cells were transfected with esiRNAs using Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778075), following manufacturer’s protocols. Transfections were performed at a final concentration of 10 pmol in a 24-well plate. Knockdown efficiency of target genes was confirmed by immunoblot analysis.
In vitro RNA synthesis
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