Vitis vinifera bud (Cabernet Sauvignon) RNA sequencing (Transcriptome study)
Total RNA extracted from Vitis vinifera bud (Cabernet Sauvignon) bud samples were used for RNA sequencing. mRNA from total RNA is enriched using oligo(dT) beads. rRNA is removed using the Ribo-Zero kit. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. The double-stranded cDNA library is completed through size selection and PCR enrichment. The libraries were sequenced using NovaSeq 6000, 150 bp Paired-end reads were generated. After series of quality control parameters, RNA-Seq reads were mapped to the grapevine reference genome PN40024 (Jaillon et al. 2007) via HISAT2 (Kim et al. 2019). The python framework HTSeq (version 0.6.1) (Anders, Pyl, and Huber 2015) was used to count the number of reads mapped to each gene. Differential gene expression and pathway analysis were performed using iDEP web application (Ge, Son, and Yao 2018).