Cloning and Characterizing miRNA Reporters

Cloning of luciferase reporters for miRNA binding sites was done as previously described^{16 (link)}. Briefly, to express different pre-miRNAs, a mammalian expression vector pBudCE4.1 was used (Thermo Fisher). PCR primers were designed to amply the genomic regions flanking pre-miRNAs (~200-nt upstream and downstream). Mouse mir124-1 was cloned downstream of the CMV promoter between Hind III and BamH I to serve as a control for transfection and expression experiments. Pre-miR-217-WT was cloned downstream of the EF-1α promoter between Not I and Kpn I. All other variants of pre-miR-217 were made using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent). All oligo sequences for cloning can be found in Supplementary Data 6. The dual-luciferase reporter assay was performed according to our published protocol^{16 (link)}, with the exception that luciferase activities were measured 48 h after cotransfection of 100 ng pre-miRNA plasmids and 100 ng corresponding reporter plasmids.