|
| Status |
Public on Nov 23, 2023 |
| Title |
24-HPI-CR2-14_TWILIGHT_Control |
| Sample type |
SRA |
| |
|
| Source name |
TWILIGHT
|
| Organism |
Pisum sativum |
| Characteristics |
cell line: TWILIGHT
cell type: leave tissue
genotype: TWILIGHT
treatment: Control
time: 24
|
| Treatment protocol |
The plants were transferred to a climate-controlled chamber one day prior to the inoculation. The climate-controlled chambers were maintained with light and dark periods of 12 hr each and a temperature of 15° C.
The plants were placed in the custom-made translucent tent fitted with pipes having holes through which misting was maintained using a humidifier. The plants were inoculated at 3-4 node stage with P. pinodes.
|
| Growth protocol |
Seeds were sown in pots prefilled with legume mix (Bio gro, Mount Gambier, South Australia, Australia) and grown in a controlled environment with light : dark ratio of 16:8 hr and day/night temperature of 24°/15° C until the plants were ready for inoculation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA from 100 mg of leaf sample was isolated using SpectrumTM Plant Total RNA kit (Sigma Aldrich, St. Louis, MO, USA) and Qiagen plant mini kit as per the manufacturer’s procedure. The quality of RNA samples was assessed by loading on to the 1% agarose gel and the concentration was checked by Nanodrop 2000 spectrophotometer (Thermal co., USA).
The total RNA extracted from each replicate was used to prepare transcriptome libraries using Agilent's Sure Select Strand Specific RNA Library Preparation Kit (Agilent Technologies) according to the manufacturer’s protocol
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw reads were processed using Fastp to remove adaptors and poly G’s and FASTQC was used to carry out quality control checks on the resulting trimmed and cleaned reads
Reads were aligned to the Pea genome reference published by Kreplak et al (2019) and quality control checks on sample variability was carried out using differential expression for sequence count data (DESeq 2) method
Read counts generated to quantify the transcripts, quantification of transcripts was carried out with a total of 1000 bootstraps to minimise effects of technical variations using Salmon as described in Li et al (2021)
Count data was then converted to a readable form using the bioconductor package Wasabi (https://github.com/COMBINE-lab/wasabi). To identify differentially expressed genes, count data generated by Wasabi was analysed using the R package Sleuth. Sleuth was used to perform likelihood ratio tests and Wald tests to detect differences that were significant between treatments (q-value < 0.05), as well as fold change approximations between treatments for abundance of each transcript respectively
Low abundance reads were removed as described before conducting DEG analyses with DEG significance defined at q-value < 0.05 and ? 1.5 for absolute fold change. We interrogated the list of DEGs generated from the above analysis for the 21 genes confirmed to be AB resistant genes from Fondevilla et al (2014) to identify the presence or absence of those genes in our list.
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample
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| |
|
| Submission date |
Jan 17, 2023 |
| Last update date |
Nov 23, 2023 |
| Contact name |
Yvonne Oby Ogaji |
| E-mail(s) |
yvonne.ogaji@agriculture.vic.gov.au
|
| Organization name |
AgriBio, Centre for AgriBioscience
|
| Department |
Department of Jobs, Precincts and Regions.
|
| Street address |
5 Ring Road
|
| City |
Bundoora |
| State/province |
VIC |
| ZIP/Postal code |
3083 |
| Country |
Australia |
| |
|
| Platform ID |
GPL32857 |
| Series (1) |
| GSE222997 |
Differential Gene Expression of Field Pea Genotypes After Peyronellaea pinodes infection |
|
| Relations |
| BioSample |
SAMN32766133 |
| SRA |
SRX19049148 |
