|
| Status |
Public on Sep 14, 2020 |
| Title |
P. syringae B728a broth rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
P. syringae B728a cells
|
| Organism |
Pseudomonas syringae pv. syringae B728a |
| Characteristics |
strain: B728a
genotype: wild-type
cell type: bacterial
|
| Growth protocol |
Bacterial cells were grown with shaking in King's medium B broth at 28°C until cultures reached a cell density of 5x10^8 cells/ml. Three replicate cultures were used. 300 µl of each culture was then applied to a 0.4 µm Isopore® membrane filter and excess liquid was removed by exposing the filter to a vacuum source for 5 seconds. Filters were then immediately placed onto King's medium B plates to incubate at 28°C for two hours while broth cultures were returned to a shaker and incubated at 28°C for two more hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells in broth cultures were harvested by pipetting 1 ml of suspension into a 15 mL conical tube containing 125 µl ice-cold EtOH/Phenol stop solution (5% water-saturated phenol (pH<7.0) in ethanol). Filters were immersed in the EtOH/Phenol stop solution and cells were harvested by sonication for 30 seconds followed by vortexing for 20 seconds to ensure complete cell detachment from the filters. Cells were then collected by centrifugation at 12,000 rpm (13,800 x g) for five minutes at 4°C. Supernatant was decanted and the cells were frozen in liquid nitrogen and stored at -80℃ until RNA isolation. RNA isolation was performed using a Direct-zol™ RNA Kit (Zymo Research). RNA was stored frozen at -80°C.
1 µl of each sample was diluted into 4 µl of RNase free water and submitted to the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley where Ribo-Zero was used for rRNA removal. RNA abundance and purity were determined using a 2100 Bioanalyzer (Agilent Technologies) and quantified using Qubit (Invitrogen). After reverse transcription, shearing of cDNA, size fractionation, and Illumina library production, the Vincent J. Coates Genomics Sequencing Laboratory samples were sequenced using an Illumina HiSeq4000 platform with 50 base pair, single-end reads. Three biological replicates were sequenced per treatment.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Reads were uploaded to Galaxy and cleaned using Trimmomatic.
Reads were aligned to the Pseudomonas syringae B728a genome using Salmon Transcript Quantification in Galaxy.
The program edgeR in R was then used to assess the statistical significance of differential gene expression. Gene expression levels were normalized using a weighted trimmed mean of M values (TMM; where M is the log expression ratio per gene between treatments).
Supplementary_files_format_and_content: Raw gene counts for every gene and sample
|
| |
|
| Submission date |
Sep 13, 2020 |
| Last update date |
Sep 14, 2020 |
| Contact name |
Monica Nicole Hernandez |
| Organization name |
Oregon State University
|
| Street address |
569 Hanley Road
|
| City |
Central Point |
| State/province |
OR |
| ZIP/Postal code |
97502 |
| Country |
USA |
| |
|
| Platform ID |
GPL29146 |
| Series (1) |
| GSE157877 |
Contact-dependent traits in Pseudomonas syringae B728a. |
|
| Relations |
| BioSample |
SAMN16121619 |
| SRA |
SRX9112742 |
