Clock Gene and Metabolic Gene Expression.

Mice were housed under LD for 4 wk; half of the animals were then exposed to DLE, while the other half remained under LD. After 2 wk of exposure to DLE, animals were killed by cervical dislocation at ZT2, ZT8, ZT14, or ZT20. Samples from the heart, liver, adrenal gland, and dorsal hippocampus (including dentate gyrus, CA1, and CA3) were collected and flash frozen on dry ice before being stored at −80 °C.

Tissue samples were homogenized in 500 μL TRIzol (Thermo Fisher Scientific) and incubated at room temperature for 5 min before adding 100 μL chloroform (Sigma Aldrich) and spinning for 15 min at 12,000 RPM at 4 °C. After phase separation, the clear lysate containing RNA was removed and further purified using the RNeasy Plus Mini Kit (Qiagen). RNA was quantified using a Nanodrop1000 (Thermo Fisher Scientific).

Complementary DNA (cDNA) was synthesized from extracted RNA using SuperScript VILO Master Mix (Thermo Fisher Scientific), adding 250 ng RNA to the reaction where possible. RT-qPCR of 11 genes of interest (Per2, Bmal1, Rev-erbα, Cry1, Dbp, Hmgcr, Hdac3, Ccrn4l, Pparγ, Npc1, and Cyp7a1) was performed using TaqMan Gene Expression Assays (Thermo Fisher Scientific) by the StepOnePlus Real-Time PCR System (Applied Biosystems); for the two housekeeping genes (Tbp and Gapdh), RT-qPCR was performed using Fast SYBR Green (Thermo Fisher Scientific). TaqMan Gene Expression Assays (Thermo Fisher Scientific) for the genes of interest were mm00478099_ml (Per2), mm00500223_ml (Bmal1), mm00520708_ml (Rev-erbα), mm00514392_ml (Cry1), mm00497539_ml (Dbp), mm01282499_m1 (Hmgcr), mm00515916_m1 (Hdac3), mm00802276_m1 (Ccrn4l), mm00440940_m1 (Pparγ), mm00435300_m1 (Npc1), and mm00484152_m1 (Cyp7a1). The primer sequences 5′ to 3′ for Tbp were TGG​GCT​TCC​CAG​CTA​AGT​TC (forward) and GGA​AAT​AAT​TCT​GGC​TCA​TAG​CTA​CTG (reverse), and for Gapdh, they were TGC​ACC​ACC​AAC​TGC​TTA​G (forward) and GATGCAGGGATGATGTTC (reverse). During RT-qPCR, each 96-well plate was loaded with cDNA samples from all four time points from each lighting condition; cDNA samples from LD and DLE conditions were amplified on separate plates.

mRNA levels of the genes of interest were quantified using the comparative threshold cycle (C_T) method (106). The 2^−CT values of these genes were expressed as a ratio relative to the geometric mean of the 2^−CT values of the housekeeping genes in the same sample (2^−ΔCT); validation of housekeeping genes is reported in SI Appendix, Supplementary Methods. Individual 2^−ΔCT values were then divided by the highest mean 2^−ΔCT value in each lighting condition to obtain 2^−ΔΔCT, indicating changes in mRNA level relative to maximal expression. In addition, CoG were obtained from the CircWave software (53–55). These values provided an estimate of the acrophase (φ) of the molecular rhythm under LD and DLE (φ_LD and φ_DLE, respectively). The extent of the phase shift, Δφ, was calculated from φ_DLE − φ_LD.