|
| Status |
Public on Jul 20, 2021 |
| Title |
HWG37_PVU_NEC_1: Common bean infected by S. sclerotiorum rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Center of ~20mm wide disease lesion on leaves
|
| Organism |
Sclerotinia sclerotiorum 1980 UF-70 |
| Characteristics |
tissue: Mycelium from leaves of 5-week old plants
treatment: in planta (Pvu)
|
| Treatment protocol |
S. sclerotiorum strain 1980 was subcultured on Potato Dextrose Agar plates at 22°C. For inoculations, a 5-mm wide agar plug containing actively growing S. sclerotiorum mycelium was placed at the center of a Petri dish or on the adaxial surface of leaves on 5-week old plants. The border (ring of ~5mm wide) and center of colonies were collected with a scalpel and frozen in liquid nitrogen.
|
| Growth protocol |
Plants were grown in Jiffy pots under controlled conditions at 23°C, with a 9 hour light period at intensity of 120 μmol/m²/s.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The edge and center of developed mycelial colonies (~20 mm diameter) was harvested and stored in liquid nitrogen. Samples were ground with metal beads (2.5 mm) in a Retschmill apparatus (24hertz for 2x1min) before solubilizing in 1mL Trizol reagent (ThermoFisher) and left for 5 min at RT. Chloroform (200 µL) was added and mixed thoroughly before incubating for 3 min at RT. After centrifugation at ~12,000g (4°C) for 15min, the upper aqueous phase was recovered and nucleic acids were precipitated by adding 2µL GlycoBlue (Ambion) and 500µL isopropanol (10 min at -20°C). After centrifugation at ~15,000g (4°C) for 15min, pellets were washed twice with 70% ethanol before drying and resuspended in RNAse-free water. To eliminate chloroform traces, water resuspended nucleic acids were further cleaned using an RNA extraction kit (Quiagen) following manufacturer’s instructions. Genomic DNA was removed by DNase treatment (TURBO DNase; Ambion) following manufacturer’s instructions. The quality and concentrations of RNAs preparations were assessed with an Agilent apparatus and chips (nano).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
HiSeq control software 2.2.58, RTA 1.18.64.0, Illumina Casava1.8.2 software used for basecalling.
Trimmomatic v0.36 was employed to trim raw reads with settings for single- or paired-end sequencing and the following settings for paired-end: ILLUMINACLIP:TruSeq2-PE.fa:5:30:10 SLIDINGWINDOW:3:18 LEADING:6 TRAILING:6 MINLEN:90
FastQC v0.11.5 (Brabham bioinformatics) was used to perform quality control of the trimmed reads. Mapping was conducted with HISAT2 (Kim et al., 2015) with --max-intronlen 500 -k 1 against the S. sclerotiorum isolate 1980 reference genome
Sorted BAM files and indices were generated with samtools v1.7
FPKM (fragments per kilobase of transcript per million mapped reads) tables were generated using the Cufflinks function cuffnorm with --compatible-hits-norm --library-norm-method classic-fpkm
Genome_build: S. sclerotiorum 1980 version 2 (GCA_001857865.1); S. trifoliorum SwB9 v1 (PRJEB36746/ERZ1667436 at https://www.ebi.ac.uk/ena)
Supplementary_files_format_and_content: tab-delimited text files include total count of uniquely mapped reads per gene for each Sample
|
| |
|
| Submission date |
Oct 21, 2020 |
| Last update date |
Jul 20, 2021 |
| Contact name |
Sylvain Raffaele |
| E-mail(s) |
[email protected]
|
| Organization name |
INRAE
|
| Lab |
LIPME
|
| Street address |
28 chemin de borde rouge
|
| City |
Castanet tolosan |
| ZIP/Postal code |
31320 |
| Country |
France |
| |
|
| Platform ID |
GPL29264 |
| Series (1) |
| GSE159792 |
Global transcriptome of the fungal pathogen Sclerotinia sclerotiorum (strain 1980) during the colonization of six plant species - Global transcriptome of S. sclerotiorum and S. trifoliorum during growth on camalexin |
|
| Relations |
| BioSample |
SAMN16510407 |
| SRA |
SRX9333866 |
