Primer probe mixes

The PureLink™ RNA mini kit from Invitrogen (Thermo Fisher) was used to purify RNA from both mock-infected and HCMV-infected LN-229 cells according to the manufacturer’s specifications. The purified RNA was re-suspended in 55 μl of RNase-free water and stored at −20 °C. SuperScript® III Platinum kit (Invitrogen by Life Technologies) was used for all qRT-PCR experiments. Custom primer-probe mixes were obtained from IDT for the following genes: IE1 forward-5′-AGCA CCCGACAGAACTCACT-3′, reverse- 5′-CAGTGCTCCCCTGATGAGAT-3′, Probe-5′-AGAGAGAGATGCCCCCGTAC-3′; IE2 forward- 5′-CAAGAGAG CGATTGGTGTTG-3′, reverse-5′−CCGCAAGAAGAGCAAAC-3′, probe-5′-TCACCTCGTCAATCTTGACG-3′.The beta actin gene was used as a housekeeping gene for normalization, and commercially available primer/probe mixes from ThermoFisher were used for amplification, according to manufacturer’s protocols. Standards were generated from purified viral DNA or from uninfected LN-229 cells. Negative (no RT) and positive controls were included on all experimental plates. All qRT-PCR experiments were done using an Analytik Jena multicolor Real-Time PCR detection system with the following one-step RT-PCR protocol: cycle 1–50 °C for 15 min (1X); cycle 2–95 °C for 2 min; and cycle 3–95 °C for 15 s (step 1) and 60 °C for 30 s (step 2) [40X].

Steady-state mRNA expression levels of eight core circadian genes in skin fibroblasts were determined by quantitative real-time PCR (RT-PCR) using Taqman MGB probes (Thermo Fisher Scientific Inc., Waltham, MA). Fibroblasts were cultured in 24-well plates and synchronized at 80% to 90% confluency by adding 100 nM dexa- methasone to the medium and incubating for 2 hr, followed by washing with DMEM (Nagoshi et al., 2004 (link)). Total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA) every 6 hr, starting at 0 −48 hr (n = 4 per time point) after the synchronization, and their quality and quantity were confirmed by NanoDrop (Thermo Fisher Scientific Inc.). The RT-PCR was performed using commercially available primer/probe mixes (Thermo Fisher Scientific Inc.) as follows: Npas2 (Mm01239312_m1), Bmal1 (= Arntl: Mm00500223_m1), Clock (Mm00455950_m1), Per1 (Mm0050 1813_m1), Per2 (Mm00478099_m1), Per3 (Mm00478120_m1), Cry1 (Mm00514392_m1), and Cry2 (Mm01331539_m1). Gapdh was used as an internal control. In addition, the LacZ reporter gene expression was determined. The statistical analysis was performed first by two-way ANOVA. The group with the significant interaction P value (P < 0.05) by two-way ANOVA and the gene expression at each time point was further subjected to the Tukey test.