Quantitative Transcriptome Analysis of Plant Samples

A Trizol kit (Invitrogen, USA) and Evo M-MLV RT Kit II (Accurate Biotechnology, AG11711, China) were used to extract total RNAs of ‘leaf’ and ‘pet’ samples, which were reverse-transcribed into cDNA. Oligo dT and random 6-mer primers were used for reverse transcription of the lncRNAs and mRNAs. Reverse transcription of the circRNAs was performed using downstream primers in qRT-PCR and random 6-mer primers. The reverse transcription of miRNA was performed using primers downstream of U6 and specific stem-loop primers. A SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) was used to perform qRT-PCR using an ABI 7300 RT-PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The protocols and conditions for qRT-PCR have been described previously, by Shi et al. [66 (link)]. The miRNA expression levels were normalized to those of U6 miRNA, which was used as the endogenous control. The expression levels of the mRNAs, lncRNAs, and miRNAs were normalized to those of actin. The primer sequences used for RT-PCR are listed in Table S8. qRT-PCRs were performed with three technical replicates and four biological replicates.