Amaxa 4d nucleofector x unit

DENV replicon plasmid (Marceau et al., 2016 (link)) was linearized using XbaI restriction enzyme. Replicon RNA was generated using the MEGAscript T7 Kit (Invitrogen) with the reaction containing 5mM m7G(5')ppp(5')G RNA Cap Structure Analog (New England BioLabs). Resulting RNA was purified by lithium chloride precipitation. For electroporation, 2 million WT or EMC4 KO HEK293FT cells were washed twice in PBS, resuspended in 100 μl SF Nucleofector solution (Lonza), mixed with 4 μg replicon RNA, transferred to a 100 ul nucleocuvette and pulsed using program CM-130 on an Amaxa 4D-Nucleofector X Unit (Lonza). Cells were then resuspended in antibiotic-free medium, distributed into 96-wells and lysed at different timepoints post-electroporation using Renilla Lysis buffer. Luminescence was measured using Renilla Luciferase Assay system (Promega) on a Spectramax i3x Multi-Mode Microplate Reader (Molecular Devices).

The Alt-R CRISPR/Cas9 system was carried out as previously described (18 (link), 19 (link)). Nrp-1 or PTEN CRISPR/Cas9 vector was transfected using an Amaxa 4D-nucleofector X unit according to the manufacturer's recommendations with program DN-100 (Lonza, Cologne, Germany).

Small interfering RNA (siRNA) for SIGNR3 was purchased from Cosmo Genetech (Seoul, Korea). Before transfection, murine non-T cells were cultured with LPS (100 ng/mL; Sigma) from Escherichia coli O111:B4. The next day, the cells were transfected using the Amaxa 4D-nucleofector X unit with a primary cell kit, as per the manufacturer’s recommendations (Lonza, Cologne, Germany).

K562 cells were split 1:5 1 day before the electroporation. Where indicated, the cells were transduced with LV or IDLV 24 h prior to electroporation. On the day of electroporation, the cells were counted on ViCell (Beckman Coulter; Brea, CA), 2 × 10⁵ cells per condition were centrifuged at 90×g for 15 min at RT, resuspended in 20 μl of SF electroporation buffer (Lonza; Basel, Switzerland), combined with Cas9 plasmid or RNP, 3 μM ssODN (where applicable), and DNA RF or GFP plasmids (where applicable). The cells were electroporated on Amaxa 4D Nucleofector X Unit (Lonza; Basel, Switzerland) using FF-120 setting. After electroporation, the cells were rested in electroporation strips for 10 min at RT, and then recovered with 500 μl of RPMI medium + 10% HI FBS (Gibco/ThermoFisher; Waltham, MA) + 1% PSQ (Gemini BioProducts; Sacramento, CA). AAV6 donor template was added to recovery medium where applicable. Twenty-four hours post electroporation, the cells were re-plated into fresh medium. The cells were harvested 4 days post electroporation for gDNA extraction to evaluate gene editing levels. gDNA was extracted using PureLink Genomic DNA Mini Kit (Invitrogen/ThermoFisher Scientific; Carlsbad, CA).