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Sample GSM6460157

Query DataSets for GSM6460157

Status

Public on Dec 18, 2022

Title

B-treated plant Rep 4

Sample type

SRA

Source name

Finely grounded frozen leaves

Organism

Vitis vinifera

Characteristics

organ: leaf
cultivar: Garnacha Blanca
treatment group: B

Treatment protocol

The treatments consisted of Akivi at 0.521 g L-1 (Aki), and Bacillus UdG at 108 CFU mL-1 lyophilized (BL) and fresh (BF). A non-treated control (NTC) was included in all the experiments. The products were sprayed on adaxial and abaxial leaf surfaces using an airbrush until near run-off. The experimental design consisted in 4 randomized blocks corresponding to the different treatment modalities (Aki, BL, BF, and NTC), each block was composed of 4 biological replicates of 5 plants.

Growth protocol

Garnacha Blanca grapevine cultivars (Vitis vinifera L.) grafted on rootstock 110R, were obtained from commercial nurserie (Agromillora Iberica, Spain). One-year-old bench-grafted grapevine rootlings were planted in a 2 L pot with 80% of the growing media (Prodeasa BV35, Burés Profesional, Spain), 20% of perlite (A-13, Agroteibe, Spain), and 4 g of the fertilizer (Osmocote® Exact Mini 3-4M, ICL Specialty Fertilizers, France). Bench-grafted grapevines were grown in a greenhouse at 25 ± 2ºC, 60 ± 10% relative humidity and a 16:8 h light:dark photoperiod. Young stocks with at least about 4 to 6 expanded leaves were used for the experiments.

Extracted molecule

total RNA

Extraction protocol

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

B_R4a

Data processing

Illumina CASAVA v1.8.2 was used for base-calling
Raw sequence files were subjected to quality control analysisis using FastQC v0.10.1
Raw sequence files were trimmed and adapters were removed with BBDuk (minimum base quality of 25 and a minimum read length of 35 bp)
Reads were mapped against the Vitis vinifera genome with STAR v2.6
Raw expression counts were obtained with FeatureCounts v1.6.1 for each annotated gene using only uniquely mapping reads (MAPQ ≥ 30).
Assembly: Vitis vinifera L. genome (Vitis vinifera cv. Pinot noir var. PN40024) (version 12X Ensembl)
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

Submission date

Aug 15, 2022

Last update date

Dec 18, 2022

Contact name

Mélina Ramos

E-mail(s)

melina.ramos66@gmail.com

Organization name

University of Girona

Lab

CIDSAV