Transcriptome Sequencing and Differential Expression Analysis in Grapevine

RNA samples were processed to construct strand-specific cDNA libraries (one per biological sample) using Illumina TruSeq RNA Library Preparation Kit (Illumina, San Diego, CA). Sequencing of all 12 libraries was conducted on a single sequencing lane using Illumina HiSeq 2000 platform (Illumina, San Diego, CA) to produce 4.2–5.2 million strand-specific 100 bp paired-end reads per library. Reads were mapped to the reference (12X) grapevine genome using STAR aligner [83 (link)] allowing only for unique mapping and up to two mismatches per read mapped, using v.2.1 gene prediction provided by CRIBI Biotechnology Center, University of Padua. Read counts were generated using HTSeq at the level of gene locus [84 ]. Analysis of differential gene expression was performed using voom/limma workflow for genes that demonstrated expression level of at least 1 count per million (CPM) in at least 4 samples [85 (link)].