Pcmv 3tag 4a

HEK293FT and Neuro-2a cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) complemented with 10% FBS, 1% penicillin–streptomycin in 5% CO₂ at 37°C. For plasmid construction, genes were cloned into pCMV-3tag-4A and pCMV-3tag-1A (Agilent Technologies). In addition, we replaced EGFP and Cas9 from pLJ-EGFP and LentiCas9-Blast (Addgene) with genes of interest. Cell transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific). shRNA clones were purchased from lentiviral vector-based shRNA libraries (MISSION shRNA library, Sigma-Aldrich). Lentivirus packaging was performed as previous reported (Shalem et al., 2014 (link)). The NMD reporter assay in N2a cells was performed as previously described (Boelz et al., 2006 (link)). Briefly, N2a cells were transfected with pCI-Renilla/β-globin and pCI-firefly reporters (gifted from Drs. Gabriele Neu-Yilik and Andreas E. Kulozik). After 24 h, cells were treated with 100 μg/mL CHX or 0.01% DMSO for 5 h. Renilla and firefly luciferase were detected using Dual-Luciferase Reporter System (Promega).