qRT-PCR Expression Analysis of DEGs

In this study, five randomly selected candidate genes for DEGs were used for RT-PCR analysis. The specific primers for these five genes and actin gene (an internal control) were designed by Primer 5.0 (Supplementary Table 2). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright^®Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States). Three biological replicates (from three independent RNA samples) were used for qRT-PCRs. For each biological replicate, three technical replicates were also conducted. The average threshold cycle (Ct) from three biological replicates was employed to calculate the gene expression fold change by the 2^–ΔΔCT method (Kong et al., 2019a (link),b (link)).