Quantitative RT-PCR Transcriptome Analysis

Total RNA was extracted from cells using Quick-RNA kit (Zymo Research) according to the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse-transcription using qScript cDNA SuperMix (QuantaBio, VWR). Real-time quantitative PCR (RT-qPCR) were performed with the Roche LightCycler® 480 instrument and the PerfeCTa SYBR Green FastMix (QuantaBio, VWR) and were carried out in a final volume of 10 μL using 0.25 µL of each primer (25 μM), 5 μL of the supplied enzyme mix, 2.5 μL of H₂O, and 2 μL of the template diluted at 1:10 [see Supplementary Table 1 for primer sequences]. After pre-incubation at 95 °C, runs corresponded to 35 cycles of 15 s each at 95 °C, 5 s at 60 °C, and 15 s at 72 °C. Melting curves of the PCR products were analyzed using LightCycler® software to exclude amplification of unspecific products. The results were normalized to different housekeeping gene transcripts (mouse RS9 or human 28S)^{[30 (link)]}.