qPCR Validation of RNA-Seq Results

For verification of RNA-Seq results, we conducted quantitative reverse transcription PCR (RT-qPCR) assays on the same RNA samples as used for RNA-Seq (RNA isolation already described above). In addition, we extended this verification to all the piglets that were sampled at the same time as the piglets used for RNA-Seq (RT-qPCR on a total of 8 pigs/group).

The total RNA was used for first-strand cDNA synthesis using Maxima H Minus First Strand cDNA synthesis kit (Thermo Fisher Scientific) according to standard procedures. All the primers (Supplementary Table 5) were designed with the software Primer3 [(61); https://primer3plus.com/primer3web/primer3web_input.htm] and pre-experimentally validated (qPCR tests including program of temperature gradient and followed by electrophoresis gel for amplicon size confirmation). All RT-qPCR reactions were conducted on the Mastercycler ep Realplex (Eppendorf) using SYBR green chemistry (Kapa SYBR Fast Universal, Sigma). The thermal cycle conditions were as follows: 1 cycle of pre-incubation at 95°C for 3 min, 40 cycles of amplification (95°C for 10 s, 60°C for 20 s, and 72°C for 20 s), and melting curve program included at the end of the run. Relative gene expression was calculated using the 2^−ΔΔCt method with the geometric mean of the Cts from the four housekeeping genes HPRT1, RPL32, GAPDH, and ACTB serving for normalization [subtracting the Ct-values of individual target genes from the Ct-value of the housekeeping genes of the same sample (ΔCt-values)]. Next, the mean ΔCt for each experimental group and target gene was calculated and subsequently used for statistical evaluation and expressing the fold change (=2^−ΔΔCt value).