Per2::LucSV knock-in ES cells (17) and Bmal1-Luc ES cells (58) were maintained on mouse embryonic fibroblast feeder cells in ES cell medium [Glasgow minimum essential medium supplemented with 15% FBS (HyClone), 0.1 mM minimum essential medium (MEM) nonessential amino acids, 0.1 mM 2-mercaptoethanol, leukemia inhibitory factor (1000 U/ml), and penicillin-streptomycin (100 U/ml)]. Per2 was disrupted using the CRISPR-Cas9 system with the target sequence of 5′-GGAACACACTGACGTCATGAGG-3′ where “AGG” is the PAM. Frameshift deletions in the targeted exon were confirmed by DNA sequencing (fig. S7) (59). For the in vitro differentiation of ES cells, cells were dissociated and seeded in low-attachment 96-well plates in differentiation medium [DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, GlutaMAX-I (Invitrogen), 0.1 mM 2-mercaptoethanol, and penicillin-streptomycin (100 U/ml)]. Two days later, the embryoid bodies were plated onto gelatin-coated 24-well plates and cultured for 26 days in differentiation medium, which was exchanged every other day. On differentiation day 28, the cells were treated with 100 nM dexamethasone for 1 hour, and then, the medium was replaced with differentiation medium containing 0.2 mM luciferin and the indicated concentrations of the compound. Luminescence was measured and integrated for 1 min at 20-min intervals with photomultiplier tube–based equipment. Period analysis was performed by sine-wave fitting, as previously described (59).

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