Real-Time Quantitative PCR Analysis

The expression levels of eight selected genes were determined using real-time quantitative PCR (RT-qPCR) to verify the expression patterns of the mRNAs identified by RNA-seq. RT-qPCR was performed on CFX Connect (Bio-Rad Laboratories Inc. Hercules, CA, United States) using HiScript II reverse transcriptase (Vazyme Biotech Co. Ltd., Nanjing, China), according to the manufacturer’s protocol. The primers used for RT-qPCR are shown in Supplementary Table 3. Sample cycle threshold (Ct) values were determined and normalized against the constitutively expressed gene (β-actin-1, internal control) (Zhao et al., 2018 (link)). The 2^–ΔΔCT method was used to calculate the relative gene expression level. Three biological replicates and three technical replicates were used for all RT-qPCR reactions.