|
| Status |
Public on Jun 06, 2017 |
| Title |
sickle Mock treated 2 dpi rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
Seedling root tissue
|
| Organism |
Medicago truncatula |
| Characteristics |
plant genotype: sickle mutant (ethylene insensitive, point mutation in A17)
days after infection/mock treatment: 2 days
disease phenotype: Highly susceptible to R. solani AG8
|
| Treatment protocol |
R. solani AG8 (isolate WAC10335) was prepared according to (Lichtenzveig et al., 2006). Seedlings were added to pots half filled with vermiculite also containing 5 ml of 2.2mg/ml dry weight equivalent suspension of homogenised fungal mycelium. The seedlings were covered with fine vermiculite and transferred to growth rooms at a constant temperature of 24 °C with 16/8 light/dark photoperiod at a light intensity of 200 mmol m-2 s-1. Mock treated plants were treated the same with the addition of 5 ml sterile water rather than the 5 ml of R. solani suspension. Inoculated plants were harvested at two and seven days after planting into pre-inoculated pots with corresponding mock samples taken at the same time. Root tissue was collected and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. All experiments included three biological replicates.
|
| Growth protocol |
M. truncatula lines A17 and skl were grown according to (Anderson et al., 2010, Plant Physiol. 154:861-873). Surface sterilised M. truncatula seeds were germinated on moist filter paper in the dark at 4 °C for two days, then one day at 24 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen root tissue was ground in the presence of liquid nitrogen and total RNA extracted using Trizol reagent (Sigma) according to manufacturer’s instructions.
Library preparation and sequencing was conducted by the Australian Genome Research Facility or Beijing Genome Institute using the TruSeq Stranded Total RNA Library Kit (Illumina)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Image analysis was performed in real time by the HiSeq Control Software (HCS) v2.2.68 and Real Time Analysis (RTA) v1.18.66.3. Then the Illumina bcl2fastq 2.17.1.14 pipeline was used to generate the sequence data
RNA-seq paired-end reads were trimmed for Illumina adapter sequences with reads trimmed to less than 25 bp discarded using Cutadapt (Martin, 2011) (parameters: --format fastq --overlap 10 --times 3 --minimum-length 25).
Trimmed RNA-seq reads were then mapped to M. truncatula genome version 4.0 (Tang et al., 2014) using Tophat2 (parameters: --b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 5000 -g 20 --report-secondary-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand) (Trapnell et al., 2009; Trapnell et al., 2012). BAM files generated from Tophat2 output were sorted using SAMtools version 0.1.19 (Li et al., 2009).
Htseq-count (Anders et al., 2015) was used to count aligned reads per gene and the resulting count matrix was normalized and analyzed for differential expression using the EdgeR package (Robinson et al., 2010) in R version 3.2.2 using a 0.05 FDR cut-off to determine differentially expressed genes.
Genome_build: Medtr version 4.0
Supplementary_files_format_and_content: Tab delimited matrix showing average log2fold change for inoculated samples over mock samples and the associated FDR from incorporation of all three replicates for each genotype/time point combination.
|
| |
|
| Submission date |
Jan 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Jonathan P Anderson |
| Organization name |
CSIRO
|
| Street address |
147 Underwood Avenue
|
| City |
Floreat |
| State/province |
Western Australia |
| ZIP/Postal code |
6023 |
| Country |
Australia |
| |
|
| Platform ID |
GPL17491 |
| Series (1) |
| GSE94260 |
Ethylene signaling is important for isoflavonoid mediated resistance to Rhizoctonia solani in roots of Medicago truncatula |
|
| Relations |
| BioSample |
SAMN06279625 |
| SRA |
SRX2527447 |
