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Sample GSM2195540

Query DataSets for GSM2195540

Status

Public on Feb 22, 2017

Title

2A3

Sample type

SRA

Source name

seedling leaf

Organism

Triticum aestivum

Characteristics

genotype: Columbus
stem rust resistance status: susceptible
tissue: seedling leaf
time point (days after after inoculation with stem rust): 0DPI
pathogen: Puccinia graminis f. sp. tritici
biological replicate: 3

Treatment protocol

Seedlings were inoculated with wheat stem rust Pgt culture 313 (pathotype 34-1,2,3,5,6,7 according to Australian nomenclature) from the Plant Breeding Institute, University of Sydney, Australia. grown in a microclimate room (22/20 °C day/night) until the two-leaf stage (approximately 2 weeks). Inoculation was undertaken by spraying the plants with urediniospores suspended in light mineral oil. Plants were dried for 30 min and incubated for 2 days in incubation cabinets (22/20 °C day/night, high humidity). Finally, plants were transferred to a microclimate room, at a lower temperature (18/17 °C day/night).

Growth protocol

Seedlings were grown in a microclimate room (22/20 °C day/night) until the two-leaf stage (approximately 2 weeks)

Extracted molecule

polyA RNA

Extraction protocol

For each sample, the first leaf of two seedlings from different pots were pooled and quickly frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Extraction Kit (Qiagen) and RNA integrity was checked on 1.4% agarose gel and on the Agilent 2100 Bioanalyzer. Poly(A) isolation was performed by the sequencing company, Australian Genome Research Facility.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed data file: DESeq_2A-2B.csv, DESeq_2A-2C.csv, DESeq_2A-4A.csv, DESeq_2A-5A.csv

Data processing

Samples were sequenced using Illumina HiSeq 2000, generating approximately 30 million 100 bp single-end reads per sample.
Sequenced reads were trimmed for adaptor sequence, masked for low-complexity or low-quality sequence, then aligned against the wheat (Triticum aestivum) Unigene database build #62 (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/unigene), comprising 158,028 transcripts, using the BioKanga software (http://code.google.com/p/biokanga). Alignment allowed a mismatch rate of up to 10% of the length of the read. Only reads with a unique best hit were selected for differential expression analysis.
Unigenes were filtered to those with a sum of counts greater than, or equal to, 100 across samples.Genes were considered expressed if more than 0.15 read per kilo base per million reads (RPKM) was detected.
Chromosomal location of each Unigene was determined using BLASTN (best hit, evalue ≤ 105) against the flow-sorted IWGSC chromosome survey sequencing contigs of cultivar Chinese Spring
Read counts were normalized and tested for differential expression, using the R DESeq package v1.12.0 with default parameters. Hierarchical clustering was done using the Euclidian distances on the normalized reads count following DESeq variance stabilizing transformation. 18 pairwise comparisons were completed (between genotypes at each time point and between time points for each genotype). Genes were considered DE for FDR ≤ 0.05 and |log2(FC)| ≥ log2(2), and for FDR ≤ 0.075 and |log2(FC)| ≥ log2(1.8) if already found DE in similar comparisons.
Genome_build: Triticum aestivum Unigene database build #62 (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/unigene), comprising 158,028 transcripts
Supplementary_files_format_and_content: Summary file with Normalised read counts for all samples, Output files from DESeq analysis

Submission date

Jun 09, 2016

Last update date

May 15, 2019

Contact name

Linda Tabe

E-mail(s)

linda.tabe@csiro.au

Phone

61262465209

Organization name

CSIRO