*3.4. Validation of RNA-Seq Data Using qRT-PCR*

To confirm the accuracy of the RNA-seq data, transcriptional levels of 14 randomly selected DEGs, representing a wide range of expression levels and patterns, were detected using qRT-PCR. All 14 DEGs participated in the process of HS response, including small heat shock protein (CA03g21390), HS transcription factor (CA03g16300), bZIP (CA08g12820), MYB (CA04g16680, CA06g27890), WRKY genes (CA03g32070, CA08g08240, and CA09g11940), NAC genes (CA05g04410, CA07g18020, CA09g12970, and CA11g04440), EG45-like domain-containing protein (CA07g00930), and universal stress protein A-like protein (CA11g00890) (Figure 3). The fold changes varied in the RNA-Seq and qRT-PCR analyses. Generally, the expression patterns determined using qRT-PCR were consistent with those obtained using RNA-Seq (Figure 3), which confirmed the accuracy of the RNA-Seq results reported in this study.

**Figure 3.** Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)-based validation of differentially expressed genes (DEGs) in response to heat stress (HS) at different time intervals. Ordinate represents fold changes of RNA-Seq data and the relative expression level of qRT-PCR. The relative expression level of each gene under stress at each time point was compared with that under normal conditions. qRT-PCR data are presented as mean ± SEM of three independent technical replicates.
