Oligonucleotides

Radiolabeled dsRNA was prepared as described previously (Iki et al. 2017 ). Briefly, 34/36-nt dsRNA was prepared from two oligonucleotides (Gene Design). The 34-nt strand was end-labeled by incubation with [γ-³²P]-dATP and T4 PNK (Thermo Scientific), whereas 36-nt strand was end-phosphorylated without radiolabeling. Single-stranded RNAs were annealed as follows: Guide and passenger strand were mixed together with 5× annealing buffer (Tris–HCl [pH 7.6] 50 mM, KCl 100 mM, and MgCl₂ 5 mM) and incubated for 2 min at 96°C in a thermomixer (Eppendorf), at which point the machine was turned off and ssRNAs were left to anneal with decreasing temperature for 12 h. 98/100- and 510/512-nt dsRNA substrates were created by in vitro transcription of individual strands using the SP6-Scribe Standard RNA IVT kit (Cellscript) with [α-³²P]-CTP, purified on mini Quick Spin RNA Columns (Roche) and extracted with phenol:chloroform:isoamyl alcohol (PCI), followed by ethanol precipitation. Annealing of single-stranded RNA to obtain 98/100- and 510/512-nt dsRNA was achieved as described for 34/36-nt dsRNA species. All three species of duplexes contain a 5′ blunt end and 2 nt 3′ overhang.