Optimized Cell Culture and Reporter Assay Protocol

Cell culture and growth conditions for HEK 293T and U2OS cells were performed as previously described [12 (link),46 (link),84 (link)]. Briefly, 293T and U2OS cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum, as previously described [84 (link),85 (link)]. For the reporter assay, 293T cells were cultured and seeded in 96-well plates, and transfected with desired plasmids using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 h later and assayed with the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase as an internal control for transfection efficiency, as detailed in our previous studies [37 (link),46 (link),86 (link)]. The luminescent CellTiter-Glo ATP assay (Promega) was used to assess cell viability. For the Lumicycle assay, samples were collected at day 6 at the end of the run when the clock phenotype was at the strongest. For the transient transfection assay, samples were collected 24 h post transfection when the Dual-Glo Luciferase Assay was performed.