Lightcycler 480 gene scanning system

To validate the accuracy of the assembly, we amplified the CDS regions of nine randomly selected transcripts from the de novo transcriptome by PCR. PCR reactions procedures followed those of Nong et al. (2019). qRT-PCR amplification was conducted using a Roche LightCycler 480 Gene Scanning system (Roche, Basel, Basel-Stadt, Switzerland) to validate the accuracy of the RNA-seq data following previously described methods [41 (link)]. The expression levels of selected DEGs were normalized by comparison with the internal reference gene UBQ [42 (link)]. The relative expression levels of each transcript were calculated using the 2^−ΔΔCt method [43 (link)]. There were three biological and three technical replicates per treatment. The primers used for PCR and qRT-PCR were designed using Primer Premier 5 (Premier Biosoft, San Francisco, CA, USA). All primers are listed in Table S1.

Total RNA was isolated from C. rosea and A. thaliana using HiPure Plant RNA Kits (Magen, Guangzhou, China), and the cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Quantitative reverse transcription (qRT)-PCR was conducted using a LightCycler^® 480 Gene Scanning system (Roche, Switzerland) and TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). Gene expression levels were normalized using the C. rosea reference gene CrEF-α as internal control. The primer pairs (CrPIP2;3RTF/CrPIP2;3RTR and CrEF-αRTF/CrEF-αRTR) used for qRT-PCR are listed in Table S3.