Mass production of Bmal1-Luc or Per2-Luc virus was performed to infect fibroblasts with the same batch of lentivirus. HEK-293 cells were seeded in HYPERflasks (Corning, Corning, New York, USA). The plasmid complex included: 60.4μg reporter plasmid; Per2 (gift from Qing-Jung Meng Lab) (37 (link)) and Bmal1 (pABpuro-BluF was a gift from Steven Brown (Addgene plasmid # 46824)), 40.25μg packaging (psPAX2 was a gift from Didier Trono Addgene plasmid # 12260), 16.1μg envelope plasmid (pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259), and 1mL of PEI-25kDa (1mg/mL; Polyscience, Warrington, Pennsylvania, USA) as the transfection reagent. Then, 16h post transfection, the cellular media (DMEM, Sigma-Aldrich, St. Louis, Missouri, USA) was replaced with fresh media and incubated at 37°C for 48h, as previously described (38 (link)). The viral supernatant was concentrated in a Vivaspin 20 and quantified using a p24 ELISA (Takara Bio, Paris, France).
Fibroblasts grown in 75cm2 flasks were transduced with a spinfection protocol. Briefly, 100 000 fibroblast cells to be transduced were lifted with TrypLE (ThermoScientific) and centrifuged with 200μl virus (~MOI 20) at 3 000g, 25°C for 99min. Cells were then resuspended in viral medium and incubated at 37°C overnight prior to use.
Fibroblasts grown in 75cm2 flasks were transduced with a spinfection protocol. Briefly, 100 000 fibroblast cells to be transduced were lifted with TrypLE (ThermoScientific) and centrifuged with 200μl virus (~MOI 20) at 3 000g, 25°C for 99min. Cells were then resuspended in viral medium and incubated at 37°C overnight prior to use.