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Sample GSM2538618

Query DataSets for GSM2538618

Status

Public on Sep 30, 2019

Title

hep1_S2

Sample type

SRA

Source name

hep1 saprophytic growth

Organism

Fusarium graminearum

Characteristics

medium: Wheat
host status: dead

Growth protocol

The highly susceptible cultivar Remus (pedigree: Sappo/Mex//Famos) (Buerstmayr et al., 2002) was used in this study. On average 20 spikelets per wheat head were inoculated by pipetting 20 000 macroconidia (10 µL of a 2* 10^6 spores/ mL suspension) on the reproductive part between the lemma and palea of the two basal florets during anthesis (resulting in 8*10^5 spores per wheat head). Mock ears were inoculated the same way, but using water instead of spore suspension. Three ears were inoculated on the living plant representing pathogenic growth of the fungus and 3 ears, which were cut off the plant and shock-frozen in liquid N prior to spore application, were inoculated as “non- response” control from the wheat side or, in other words, representing saprophytic growth of the fungus. After inoculation the wheat heads on the living plants were covered with moistened plastic bags for the first 24 hours to provide high humidity. The inoculated dead wheat heads were placed in glass petri dishes (Ø 140 mm; h 20 mm). Incubation conditions were set at 20°C, 50% relative humidity during daytime and 18°C, 50% humidity during night with a 16 h photoperiod. Harvesting was performed 3 dai and 5 dai by freezing the plant material in liquid nitrogen. Only palea and lemma of the inoculated florets were sampled including the respective part of the rachis. Additionally completely untreated wheat heads were sampled.

Extracted molecule

total RNA

Extraction protocol

RNA isolation was performed using RNeasy Plant Mini Kit (Qiagen, 74904) following the instructions of the manufacturer with one modification and one extension: Instead of eluting RNA once in 30 µL RNase- free water, elution was performed in 2x 20 µL RNase- free water with a 3 minute standing period at room temperature after each water application to the column. Additionally on-column DNase digestion was done during the extraction protocol by using RNase-Free DNase Set (Qiagen, 79254). RNA quantity was first checked on the NanoDrop 2000c Spectrophotometer (Thermo Scientific). Afterwards RNA quantity and integrity was validated by using the Agilent RNA 6000 Nano Kit (Agilent Technologies, 5067-1511) on an Agilent 2100 Bioanalyzer machine following the instructions of the provider. The cDNA Synthesis was done with the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, # K1632) using random hexamer primer and following the manufacturer´s protocol.
Illumina sequencing libraries were made from RNA samples according to TruSeq RNA Sample prep kit v2 (Illumina) following the manufacturers protocol with 1 µg total RNA input. 50 bp single end sequencing was performed using a HiSeq Illumina sequencer. Obtained sequences were de-multiplexed, quality controlled and mapped on the Fusarium graminearum PH-1 genome assembly (p3_p13839_Fus_grami_v32).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

To analyse the transcriptomes within pathogenic and saprophytic Fusarium graminearum-Triticum aestivum interactions, as well as axenic F. graminearum cultivations, the RNA-seq reads were mapped on the reference genome of Fusarium graminearum or Triticum aestivum using TopHat (v2.0.8) (Trapnell et al., 2012). The interval for allowed intron length was set to a minimum of 20 nt and maximum of 1 kb (Trapnell et al., 2009, Kim et al., 2013).
We used Cufflinks to determine the abundance of transcripts in FPKM (Fragments Per Kilobase of exon per Million fragments mapped) and calculated differentially expressed genes using Cuffdiff (Trapnell et al., 2010, Trapnell et al., 2012). The gene models were included as raw junctions. Transcripts with a FPKM count of zero in one condition were set to the minimum global FPKM count of both conditions.
Genome_build: The reference genome for mapping the F. graminearum reads was retrieved from Wong et al. 2011 (PMID:21051345). To map the T. aestivum reads we used the reference genome given by Mayer et al. 2014 (PMID:25035500).
Supplementary_files_format_and_content: GSE96705_hep1_raw_fpkm.txt
Supplementary_files_format_and_content: GSE96705_wheat_raw_fpkm.txt (abundance data derived from "mixed read" files; see file headers for GEO Sample numbers).

Submission date

Mar 16, 2017

Last update date

Sep 30, 2019

Contact name

Stefan Boedi

E-mail(s)

stefan.boedi@boku.ac.at

Organization name

BOKU

Street address

Konrad Lorenzstr. 24