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Sample GSM7056540

Query DataSets for GSM7056540

Status

Public on Jun 06, 2023

Title

YoGI_026 after_drought_stress

Sample type

SRA

Source name

Leaves

Organism

Triticum aestivum

Characteristics

tissue: Leaves
genotype: YoGI_026=CIMMYT collection accession identifier CWI 6118
treatment: drought

Treatment protocol

Four plants of each accession were watered normally to maintain soil moisture, until they reached GS13 (Zadok’s growth scale) whereby stress was applied by withholding water for a ten-day period. Normal watering then resumed for three-days to serve as a recovery period. The negative control consisted in four replicates of each accession growing at the same time but not being exposed to drought stress. Plants were harvested 13 days after GS13.

Growth protocol

Seeds fo the 14 wheat landraces were sown in Levington Advance Seed & Modular F2S compost mixed with Aggregate Industries Garside Sands 16/30 sand (80:20 ratio). The pots were treated with CaLypso insecticide (Bayer CropScience Ltd., 0.083ml mixed with 100ml water, applied to each litre of compost) and grown under long day (16/8h, 20°C/14°C) glasshouse conditions.

Extracted molecule

total RNA

Extraction protocol

Each RNA extraction consisted in 6cm of leaf tissue. The before treatment were the wheat seedlings upon reaching GS13 whilst the after treatment was collected at the end of the drought period (13 days after GS13, 10 days draught stress+3 days recovery). Tissue was collected individually for each sample, and immediately immersed in liquid nitrogen to prevent nucleic acid degradation. Tissue samples were stored at -80°c for later processing. Total RNA was extracted from <100 mg of individual leaf tissue samples using the E.Z.N.A Plant RNA Kit (Omega Bio-Tek, GA, USA) including a DNase treatment, according to the manufacturer’s protocol.RNA was measured using NanoDrop ND-1000 Spectrophotometer (Thermo-Fisher Scientific, MA, USA) and a Qubit 4 Fluorometer (Life Technologies, CA, USA) whilst the quality was assessed by running 2 μl of sample on an Agilent Technology 2100 Bioanalyzer and only keeping samples with RIN>7. Replicates (4 plants) were pooled into 1 sample per landrace per treatment at equimolar proportions.
The library was obtained using TruSeq Stranded mRNA (Illumina). The mRNA was purified using poly-T oligo attached magnetic beads, fragmented and the first strand cDNA synthesized using random hexamer primers, whilst the second strand cDNA using dUTP for directional library. End-repair, A-tailing, adapter ligation, size selection,USER enzyme digestion to remove the UTP-containing second strand cDNA, PCR amplification and purification
TruSeq stranded mRNA run in the Illumina Novaseq 6000 platform (Illumina, CA, USA) with an 150bp paired-end sequencing strategy

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Column A_YoGI_026 in processed files

Data processing

After sequencing, quality control was carried out using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw reads were then filtered by trimming low quality sequences (average Phred score < 15), trimming short length reads (<36bp), and clipping Illumina adapters using Trimmomatic v0.39
Salmon was used to map reads to the Triticum aestivum reference genome v1.1 (IWGSC RefSeq v1.1). Salmon’s mapping-based mode was used to create an index from the reference genome, and then for quantification of the trimmed reads. Salmon output files were prepared for differential expression analysis using the R package TxImport, generating a table containing transcript abundance, counts, and length from the Salmon quantification files
Assembly: IWGSC V1.1 reference (https://wheat-urgi.versailles.inra.fr/Seq-Repository/Assemblies)
Supplementary files format and content: Salmon output

Submission date

Feb 22, 2023

Last update date

Jun 06, 2023

Contact name

Sara Franco Ortega

E-mail(s)

sara.francoortega@york.ac.uk

Organization name

Centre for Novel Agricultural Products, University of York

Department

Department of Biology