0.5 µg total RNA was reverse-transcribed by RevertAid™ first strand cDNA synthesis kit using oligo-dT primer (Thermo Fisher Scientific). We assessed expression of 23 genes from the following categories: cytokines (IFNB1, IL1B, IL6, IL10, IL12B, IL23A, TNF), interferon response markers (MX1), NOD1 receptor (NOD1) and its adapter (RIPK2 [RIP2]), NF-κB family transcription factors (TFs) (NFKB1 [NFKB-p50], RELA [p65]), negative regulators of NF-κB signaling (NFKBIA [IKBA], TNFAIP3 [A20]), NT genes from (13 (link)) (FPR1, PTGES), E2F family TFs (E2F1, E2F2), and several genes classified as hyperinducible upon LPS ➔ LPS sequence in our RNA-seq experiments (WNT5A, PIM2, PKIG, RIPOR2). Amplifications were performed using 7300 Real Time PCR System (Applied Biosystems) under conditions described earlier (30 (link), 34 (link)). Sequences of primers are listed in Supplementary Table S1. Relative expression (RE) was calculated by the 2–ΔΔCt method using non-stimulated macrophages from each donor as reference samples and GAPDH as the house-keeping gene. Two-times or higher differences of RE were considered biologically significant.
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