Drug inventory, purified enzymes and the synthesis of double-stranded RNA

Following drugs were dissolved in nuclease-free water, kept at −20°C and injected at the needle concentration of: Cycloheximide (50ug/mL; Sigma-Aldrich), Colchicine (100ug/mL; Sigma-Aldrich), Rhosin Hydrochloride (19.6mg/mL; R&D Systems) and Y-27632 (76mM; Adipogen). Following drugs were dissolved in DMSO (ranging from 50–100% in stock solution), kept at −20°C, and injected at the needle concentration of: Cytochalasin B (11.7mg/mL; ACROS Organics), Roscovitine (10mM; SelleckChem) and Nocodazole (10mg/mL; Sigma Aldrich). Purified Exoenzyme C3 transferase protein (Cytoskeleton Inc) was prepared with nuclease free water, kept at −80°C and injected at a needle concentration of 100ug/uL.

dsRNAs were synthesized by RNA Greentech LLC (Texas, USA) and stored at −80°C. Coding sequences used to generate the dsRNAs are listed in key resources table. To cross-confirm the validity of the correct RNA product in house, the dsRNAs were ran on electrophoresis (1.5% agarose gel) using 2xRNA loading buffer (ThermoFisher Scientific). For electrophoresis, a 1:1 mixture of loading buffer and dsRNA was heated to 65°C for 5 min and then was transferred to ice to decompose any secondary structure on the dsRNA.