Sub9kits

The dicer siRNA substrates targeting the USP14 gene obtained from IDT (Coralville, Iowa), were encapsulated into lipid nanoparticles using microfluidic technology by Precision NanoSystems (SUB9KITS™; Vancouver, Canada). Cells were treated with 0.1 μg/ml of Neuro9™ RNAi nanoparticles as described in [68 (link)] for 72 h prior to treatment with 10 μM PGJ2 for 16 h. During siRNA treatment, cells remained in Neurobasal media supplemented with 2% B27 and 0.5 mM L-Glutamax (all from Invitrogen) at 37°C in a humidified atmosphere containing 5% CO₂. Following the 72 h incubation period with siRNA, the medium was changed to DMEM without serum, supplemented with 0.5 mM L-Glutamax and 1 mM sodium pyruvate (all from Invitrogen) and cells were treated with 10 μM PGJ2 for 16h.

The LNP-siRNA was formulated using a proprietary mixture of lipids containing an ionizable cationic lipid, supplied as SUB9KITS™, and the siRNA was encapsulated using a microfluidic system for controlled mixing conditions on the NanoAssemblr™ instrument (Precision Nanosystems, Vancouver, Canada) [12 (link)]. Concentrations in our manuscript always refer to siRNA (1μg/ml signifies 1μg of siRNA in 1ml of culture and 5mg/kg signifies 5mg of siRNA per kg of body weight) (0.25, 0.5, 1 and 2 μg/ml siRNA, corresponding to 17.85, 35.7, 71.43, and 143 nM siRNA, respectively). Further details about composition and properties of formulations are provided in supplementary methods.