qRT-PCR Validation of RNA-Seq Differentially Expressed Genes

qRT-PCR was used to confirm the accuracy of the differential expression of DEGs via RNA-Seq. Total RNA was isolated from the leaf samples and treated with DNase I to remove any genomic DNA contamination. The single-stranded cDNAs used for real-time PCR analysis were synthesized using a PrimeScript^TM RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was carried out using SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China) on an Eppendorf Real-Time PCR System (Mastercycler® ep realplex, Germany) according to the manufacturer's protocol, and the amplification was conducted as described by Ren et al. (2014 (link)). The C. sinensis β-actin gene (Csβ-actin, GenBank: HQ420251.1) was amplified as an internal reference standard, and the relative expression levels were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Three biological and three technical replicates were performed for each sample, and the primers used for qRT-PCR are listed in Supplementary S1.