Polycistronic anti-ChAT iRNA Sequences

The polycistronic anti-ChAT iRNA sequences were identified by the DSIR algorithm (Supplementary Fig. 1b). The same elimination steps discussed above were followed and the four most highly ranked sequences were selected and modified to mimic the endogenous mir17-19b [17 (link)]. Complete Scaffolds flanked by overlapping sequences of the pLenti-syn::hM4Di-CFP plasmid [3 (link)], including restriction site BsRGI and AscI flanking the 5′ and SalI flanking the 3′ end to offer the option of either restriction or In-Fusion cloning (Clontech). The DNA was synthesized and cloned into a pUC57 vector by Genewiz (https://www.genewiz.com). In-Fusion cloning (Clontech) was used to place scaffolds 3′ of the hM4Di-CFP open-reading frame of the pLenti-syn::hM4Di-CFP plasmid, digested with AscI and SalI. MirE1 and MirP constructs were also cloned into the pLenti-syn::mCherry plasmid in which the hM4Di-CFP open-reading frame was replaced by the mCherry open-reading frame via gene synthesis (Genwiz) and in fusion cloning. BrGI was used instead of AscI for digesting the plasmid before Infusion cloning (Clontech).