Rhythmic Gene Expression Analysis in Neural Cells

For gene expression analyses, NPCs (n=2 control, 2 Li-R, 2 Li-NR) or neurons (n=2 control, 2 Li-R, 3 Li-NR) were grown to a density of 400×10³/well. Cells were synchronized using 10 μM forskolin (Tocris). After 16 h, plates were collected at 6 h intervals over 24 h. Plates were washed with cold PBS and frozen at −80°C. RNA was extracted using RNeasy kit (Qiagen) and quantified by spectrophotometer. RNA (500 ng) was reverse transcribed into cDNA using QuantiTect RT-PCR kit (Qiagen). RT-PCR was performed on a BioRad CFX384 thermocycler using pre-validated Taqman primers (Applied Biosystems) for BMAL1 (ARNTL), CLOCK, CRY1, NR1D1, PER1/2/3, and RORA. Expression was normalized to GAPDH, a non-rhythmic reference gene^{18 (link)} and normalized to mean expression over 24 h. Using data matched for sample and experiment, correlation coefficient across time for each gene pair were calculated to identify network-level patterns in expression. Correlation coefficients were z-transformed and network connectivity was visualized using Cytoscape^{19 (link)} and analyzed by 2-way ANOVA.