2.6. Real-time quantitative PCR

Total RNA was extracted from FBS-MDMs stimulated or not with 25 ng/ml IFN-β, IFN-γ, IFN-λ1, or IL-27 (BioLegend) for 18 h, as reported in section 2.2. Then, 300 ng of RNA was used to construct cDNA libraries for each experimental group using the commercial RevertAid Minus First Strand cDNA Synthesis Kit (Thermo Scientific), following the manufacturer´s protocol. RT-qPCR was used to validate the expression of 14 key DEGs obtained by RNA-Seq using a set of specific primers ( Supplementary Table 1 ). PCR amplifications were performed using the Maxima SyBR Green system (Thermo Fisher Scientific). The Bio-Rad CFX manager was used to obtain the cycle thresholds (Ct) determined for each sample using a regression fit in the linear phase of the PCR amplification curve. The relative expression of each target gene was normalized to that of the unstimulated control and housekeeping gene GAPDH (ΔΔCt) and was reported as the Log2FC ( Supplementary Table 2 ).