Genome-length Jad cDNA Transcription and Transfection

Genome-length Jad and intergenotypic Jad/C recombinant cDNAs were linearized with XbaI and treated with mung bean nuclease (New England BioLabs, Evry, France) prior to in vitro transcription using T7 RiboMAX Express Large Scale RNA production system (Promega) and purification of resulting synthetic RNAs, as described previously^{49 (link)}. Huh-7.5 cells (2 × 10⁶ cells) were transfected by electroporation with 5 µg of synthetic, genome-length RNAs, in 4-mm-gap-width cuvettes by applying one pulse at 240 V at 900 F using EasyjecT Plus instrument (Equibio, Lancashire, United Kingdom). Electroporated cells were then immediately resuspended in complete medium and seeded at 1.6 × 10⁶ cells per 75 cm² flask.