Circadian clock gene expression analysis

RNA isolation and cDNA synthesis was performed according to a previously established method [75 (link)]. Following tissue collection of the NAc, SCN, and VTA, RNA was isolated in Purezol reagent and collected using the Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue Kit. RNA was reverse transcribed into cDNA using the Bio-Rad iScript cDNA Synthesis Kit. RT-qPCR reactions were carried out in duplicate with negative controls (primer only controls, and no reverse transcriptase sample controls) using the Bio-Rad SYBER Green Supermix (Bio-Rad, Hercules, CA, USA), gene-specific primers, and the Bio-Rad CFX384 Real-Time System. Established primers targeting Clock, Npas2, Bmal1, Per1, Per2, Per3, and 18s (primer sequences provided in Table 2) [9 (link),12 (link),80 (link)].