Primescript rt enzyme with a gdna eraser

Total RNA was isolated and purified according to previous methods using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) [57 (link)]. To ensure the accuracy of the target genes, quantitative real-time PCR (qRT-PCR) analyses were carried out to validate the expression of various catechins biosynthetic genes. An aliquot of 1 µg of total RNA was converted to first-strand cDNA using a PrimeScript RT enzyme with a gDNA eraser (Takara, Japan). The CDS (coding sequence) generated from the reference genome was downloaded from TPIA database (NCBI accession number PRJNA597714) for the primer design. The primers for qRT-PCR were designed by Primer3Plus (https://primer3plus.com/, accessed on 20 January 2021) and listed in Table S4. The qRT-PCR was performed on an CFX96 Real-Time PCR Detection System (Bio-Rad, Foster City, CA, USA) using SYBR Premix Ex Taq II (Takara, Japan). Next, the transcript levels of the target genes were monitored with 18s rRNA as the internal control for normalization and calculated using the 2^−∆∆ct method [58 (link)]. Moreover, ΔCt variation analyses at different template concentrations were performed for each primer pairs to validate the 2^−∆∆ct method, and the relative ΔCt equations were listed in Table S4 [58 (link),59 (link)]. All these experiments were performed with three biological replicates.

For RNA-Seq validation, 1 µg of total RNA used in the previous RNA-Seq library construction was used for cDNA synthesis. For the candidate gene expression analysis, the cDNA was synthesized from 1 μg of total RNA for each sample. Each sample was collected from nine individual buds. A PrimeScript RT enzyme with a gDNA eraser (Takara, Japan) was used for cDNA synthesis. qRT-PCR was performed on an Applied CFX96 Real-Time PCR Detection System (Bio-Rad) using SYBR^®Premix Ex Taq™ II (Takara, Tokyo, Japan). Primers designed from the conserved region of each cDNA were used for the qRT-PCR analyses (Tables S4 and S5). C. appendiculata actin (CaActin, Cluster-32503.44149) and elongation factor 1 α (CaEf-1α, Cluster-26967.95811) were used as the internal reference controls. The analysis was performed with three biological replicates. Relative expression levels (compared with 0 days) were calculated using the standard 2^−ΔΔCt method.