Hiscript rt supermix for qpcr gdna wiper kit

The RT-qPCR test was carried out to quantify gene expression levels [28 (link),29 (link)]. The TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract total RNA from S. Enteritidis cells cultured as described in Section 2.3. The cDNA was then synthesized using a HiScript RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme Biotech Co., Ltd., Nanjing, China). Primers in Table 1 were referenced from published data [14 (link),15 (link)]. PCR amplification was initiated at 95 °C for 5 min, followed by 40 cycles at 95 °C for 5 s, 55 °C for 15 s, and 68 °C for 30 s. Change in gene expression levels of S. Enteritidis under ethanol stress was compared with that in pure TSB-YE with 16S rRNA as the reference gene by the 2^−ΔΔCt method [30 (link)].

For cDNA synthesis, 1 μg total RNA was processed using the HiScript RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme). The ChamQ Universal SYBR qPCR Master Mix (Vazyme) was used for the thermocycling reaction. The RT‐qPCR analysis was carried out in triplicate times. Primer sequences were as follows:

Beta‐ACTIN: Forward: 5′‐GTGGCCGAGGACTTTGATTG‐3′,

Reverse: 5′‐CCTGTAACAACGCATCTCATATT‐3′,

CXCL11: Forward: 5′‐GACGCTGTCTTTGCATAGGC‐3′,

Reverse: 5′‐GGATTTAGGCATCGTTGTCCTTT‐3′.