Plightswitch 5utr

To establish whether the non-coding exons of the isoforms expressed in the dorsal skin of Peromyscus showed differences in translation, we generated luciferase reporter plasmids by cloning each of the three noncoding exons (1C, 1D, or 1E) from both P. maniculatus wideband and wild-type strains into a 5' UTR reporter vector (pLightSwitch_5UTR; Switchgear Genomics) upstream of the luciferase coding sequence and downstream of the ACTB promoter. We generated mutated 1D reporter plasmids from the wild-type 1D plasmid using the Q5 Site-Directed Mutagenesis Kit (New England BioLabs). We transfected plasmids into HEK293 cells using Lipofectamine (Invitrogen), with six replicate transfections per construct. Forty-eight hours after transfection, we measured luminescence as a readout of protein production using a microplate reader (SpectraMax L). We quantified transcription from each plasmid as follows: we collected six replicates of transfected cells in TRIzol reagent (Invitrogen) and extracted RNA using the Direct-zol RNA Mini Kit (Zymo Research). We then carried out qPCR as described above using the primer pairs Luciferase-F and -R, and β-actin-Human-F and -R. Primer sequences can be found in Supplementary Table 1.