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Sample GSM2214215

Query DataSets for GSM2214215

Status

Public on Feb 10, 2017

Title

3A-144-R1

Sample type

SRA

Source name

Infected plant tissue_3A-144

Organism

Fusarium graminearum

Characteristics

experiment type: in-planta inoculations
infection in: spring wheat cultivar- Briggs
inoculated with: 3ADON isolate population
tissue: Inoculated Spikelet
time point: 144 HAI
sequence id: 9248x5

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Promega SV total RNA isolation kit. Extracted RNA was converted dscDNA and used for library preparation and sequencing using TruSeq RNA sample preparation kit from Illumina.
The library was constructed using TruSeq RNA sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Briefly, the poly-A containing mRNA was purified from the total RNA using the poly-T oligo-attached to the magnetic beads. After purifications, mRNA was fragmented into small pieces using divalent cations under elevated temperature. The fragmented mRNA was converted to first strand cDNA using reverse transcriptase and random primers. Single strand cDNA was further converted to double strand (ds) cDNA using DNA polymerase I and RNase H. Then, the ds cDNA fragments were end repaired, ligated with indexing adapters, purified and enriched with PCR to develop the library. The prepared libraries were sent to Huntsman Cancer Institute, the University of Utah (Salt Lake City, UT) for generating 50bp single-end reads with the Illumina HiSeq 2000 sequencing system

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Mapping of sequence reads to reference genome, and analyses of transcript abundance and differential gene expression were performed as described by Trapnell et al. (2012).
The trimmed reads (37 bases) were then aligned to the F. graminearum reference genome downloaded from the Broad using Bowtie v0.12.5 and TopHat v2.0. with default settings. Cufflinks v0.9.3 was used to calculate transcript abundance based on fragments per kilobase of transcript per million fragments mapped (FPKM) using all parameters on default settings.
The pairwise comparisons of gene expression profiles between the two populations were done using the Cuffdiff program of the Cufflinks version 1.3.0 (Trapnell et al, 2010).
Principal component analysis (PCA) was performed using JMP Genomics v 6.0 (SAS Institute Inc., Cary, NC) for all genes (except novel transcripts).
The differently expressed genes (DEGs) were functionally categorized online for all pairwise comparisons according to the Munich Information Center for Protein Sequences (MIPS) functional catalogue (Ruepp et al. 2004). The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed using interface on Blast2GO.
Genome_build: The trimmed reads (37 bases) were then aligned to the F. graminearum reference genome downloaded from the Broad Institute.
Supplementary_files_format_and_content: File includes total number of reads, number of reads mapped to the reference genome, differently expressed genes in all pairwise comparisons.

Submission date

Jun 27, 2016

Last update date

May 15, 2019

Contact name

Shaobin Zhong

E-mail(s)

shaobin.zhong@ndsu.edu

Phone

701-231-7427

Organization name

North Dakota State University