Cfx96 real time system pcr instrument

Total RNA was extracted from cells in each treatment group using an RNA Fastagen Kit and reverse-transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (+ gDNAwiper). RT-qPCR was performed using ChamQ Universal SYBR qPCR master mix kit and specific primers for glycerol 3-phosphate dehydrogenase (GAPDH), IL-1β, and IL-8 in a CFX96™ Real-time System PCR instrument (Bio-Rad, CA, USA), with GAPDH as the reference gene. The relative mRNA expression levels of IL-1β and IL-8 were calculated using the 2-△△CT method. The CT value of the PRRSV-JXA1 Nsp9 gene was detected using qRT-PCR with AceQ Universal U+Probe master mix V2 and JXA1 Nsp9 specific primers and probes. All experiments were performed in triplicate. The sequences of the gene-specific primers and probes are listed in Table 1.Table 1

RT-qPCR primer and probe sequences.

Table 1

Gene	Primer sequence (5 '−3′)
IL-1β-F	GGAAGACAAATTGCATGG
IL-1β-R	CCCAACTGGTACATCAGCAC
IL-8-F	AGGACAAGAGCCAGGAAG
IL-8-R	CTGCACCTTCACACAGAGC
Nsp9-F	CCTGCAATTGTCCGCTGGTTTG
Nsp9-R	GACGACAGGCCACCTCTCTTAG
JXA1-Nsp9-Probe	ACTGCTGCCACGACTTACTGGTCACGCAGT
GAPDH-F	CCTTCCGTGTCCCTACTGCCAAC
GAPDH-R	GACGCCTGCTTCACCACCTTCT

Gene expression analysis was carried out with the 2X SYBR Green Fast qPCR Mix Kit (RK21204, ABclonal Corporation). qPCR reactions included 1 μL cDNA, 10 μL 2X Supermix, 0.8 μL Primer mix, and 8.2 μL ddH2O. Amplification was performed on a CFX96 Real-Time System PCR instrument (Bio-Rad) and fluorescence values were collected. The relative expression levels of genes were calculated using the 2ˆ(-ΔΔCt) method with TaActin as the internal reference gene. For ChIP-qPCR assay, input DNA and immunoprecipitated DNA were used simultaneously for qPCR detection, with input DNA serving as control. The primers used for qPCR and ChIP-qPCR are listed in Table S6.