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Status |
Public on Sep 30, 2019 |
Title |
hep1_S3 |
Sample type |
SRA |
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Source name |
hep1 saprophytic growth
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Organism |
Fusarium graminearum |
Characteristics |
medium: Wheat host status: dead
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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.
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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).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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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).
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Submission date |
Mar 16, 2017 |
Last update date |
Sep 30, 2019 |
Contact name |
Stefan Boedi |
E-mail(s) |
stefan.boedi@boku.ac.at
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Organization name |
BOKU
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Street address |
Konrad Lorenzstr. 24
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City |
Tulln/Donau |
ZIP/Postal code |
A-3430 |
Country |
Austria |
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Platform ID |
GPL17573 |
Series (1) |
GSE96705 |
Loss of heterochromatin protein 1 (Hep1) in Fusarium graminearum leads to hypervirulence and mycotoxin overproduction during pathogenic growth. |
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Relations |
BioSample |
SAMN06607521 |
SRA |
SRX2646763 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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