GSM6460162: Non-treated plant Rep 1; Vitis vinifera; RNA-Seq
Sampling was carried out 24 h after spraying plants with the products. Four biological replicates were sampled for each treatment for RNA-sequencing analysis (RNA-seq), and three biological replicates for reverse transcription quantitative PCR (RT-qPCR) analysis. Two leaves from each plant (5 plants per repetition) were harvested, grounded, and soaked in liquid nitrogen. Each ground leaf sample was added to 2 mL tubes containing two borosilicate glass beads in order to obtain a fine powder using Tissuelyzer II system (Qiagen, USA) for 1 min at 30 Hz. For total RNA isolation from grapevine leaves, the commercial kit SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA) was used  following manufacturer's instructions. Residual DNA was removed using Invitrogen™ TURBO DNA-free™ Kit (Applied Biosystems, USA). The RNA-seq transcriptome library was prepared using the TruSeq Stranded mRNA Sample Prep kit (Illumina, USA) following the manufacturer's instructions using 1-2 µg of good quality RNA (R.I.N. > 7) as input. The RNA was fragmented 3 minutes at 94°C and every purification step was performed by using 0.81X Agencourt AMPure XP beads. Final libraries were quantified by using the Qubit 2.0 Fluorometer (Invitrogen, USA) and quality tested by Agilent 2100 Bioanalyzer RNA Nano assay (Agilent technologies, USA). Libraries were then processed with Illumina cBot for cluster generation on the flowcell, following the manufacturer's instructions and sequenced on paired-end (2x150 bp, 30M reads per sample) at the multiplexing level requested on NovaSeq6000 (Illumina). The CASAVA 1.8.2 version of the Illumina pipeline was used to processed raw data for both format conversion and de-multiplexing.