|
| Status |
Public on Feb 03, 2018 |
| Title |
Mock 1hpi Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Mock-treated Arabidopsis leaves
|
| Organism |
Pseudomonas syringae pv. tomato str. DC3000 |
| Characteristics |
strain: DC3000
time of collection: 1 hour post inoculation
host plant tissue: leaves
host plant treatment: mock
time post infection: 1 hour
|
| Treatment protocol |
Four marked leaves each on plants (24-31 per time point per treatment) were treated with either 1 mM flg22 in 0.1% DMSO or with 0.1% DMSO (mock) by infiltration with a 1 mL blunt syringe on the abaxial surface. Plants were kept uncovered in the growth room for 20 hours prior to bacterial inoculation.
|
| Growth protocol |
Pseudomonas syringae pv. tomato DC3000 was streaked from a single colony onto King’s B media and grown overnight at room temperature. Bacterial inoculum was smeared into a lawn using liquid media and incubated overnight at room temperature. For individual inoculations, bacteria were harvested from plates, suspended in 0.25 mM MgCl2, and diluted to an OD600 of 0.8 (~1X10^9 CFU/mL)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Leaves from each treatment were arranged on two sheets of parafilm, rolled and inserted into the barrel of a two 20 mL syringes. A RNAlater-like RNA stabilizing buffer pH 5.2, was poured into each syringe, sealed, and vacuum infiltrated at 95 kPa for 2 min followed by a slow release of the vacuum. Vacuum-infiltration with RNA stabilizing buffer was conducted twice on inoculated leaves. Excess RNA stabilizing buffer was decanted and the syringes were placed into 50 mL conical tubes and centrifuged at 1000 g for 10 min at 4°C to recover the intercellular wash fluid. The flow-through was pooled for each treatment and concentrated by syringe filtration using a 0.20 µm Micropore Express Plus membrane placed within a removable filtering syringe tip adapter. Filters were placed in homogenization tubes and flash frozen in liquid nitrogen. The filter membranes were homogenized in Trizol Reagent for 1 min at 1750Hz using a GenoGrinder followed by the Direct-Zol Miniprep Plus kits, which included an on-column DNAse I treatment (30 U/column for 15 minutes). RNA samples were additionally treated with TURBO DNase to eliminate DNA contamination. Total RNA underwent RiboZero treatment to remove bacterial rRNA.
Illumina TruSeq Stranded Total RNA Sample Prep Kit was used for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
processed files .rpkm_0, .rpkm_1, and .rpkm_1 correspond to abundance measurments for NC_004278.1, NC_004632.1, and NC_004633.1 respectively
|
| Data processing |
Illumina BaseSpace Sequence Hub used for base-calling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using Trimmomatic v.032
Trimmed reads were aligned to the P. syringae pv. tomato DC3000 reference genome (NC_004278.1, NC_004632.1, NC_004633.1) using EDGE-pro v.1.3.1
Counts of RNA-Seq fragments were computed for each annotated gene from RPKM values using the egdeToDeseq.perl script within EDGE-pro.
Genome_build: NC_004278.1, NC_004632.1, NC_004633.1
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each gene for each sample
|
| |
|
| Submission date |
Feb 02, 2018 |
| Last update date |
Feb 05, 2018 |
| Contact name |
Brian H Kvitko |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Georgia
|
| Department |
Plant Pathology
|
| Street address |
120 Carlton St
|
| City |
Athens |
| State/province |
GA - Georgia |
| ZIP/Postal code |
30602 |
| Country |
USA |
| |
|
| Platform ID |
GPL24578 |
| Series (1) |
| GSE110100 |
In planta transcriptome analysis of Pseudomonas syringae pv. tomato DC3000 during early infection and early exposure to pattern-triggered immunity in Arabidopsis thaliana |
|
| Relations |
| BioSample |
SAMN08457937 |
| SRA |
SRX3644658 |
