|
| Status |
Public on Dec 31, 2023 |
| Title |
PA_in_CAA_FeHigh_2 |
| Sample type |
SRA |
| |
|
| Source name |
PAO1 grown in 100% CAA medium_high iron_replicate 2
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
strain: wt
growth phase: planktonic cells, mid to late exponential phase
medium: 100% CAA medium
iron availability: high, 20 uM FeCl3 (no Transferrin)
|
| Treatment protocol |
We generated 3 replicates per treatment. PAO1 was grown in either 100% fresh CAA medium only, in a mixture of 70% fresh CAA medium with 30% PAO1 supernatent or in 70% fresh CAA medium with 30% Burkholderia cenocepacia H111 supernatent. These treatments were each conducted under iron limited and iron rich conditions.
|
| Growth protocol |
We first prepared two starter cultures of PA by inoculating 20 ml LB medium with 200 µl of a PA overnight culture in 100 ml Erlenmeyer. These starter cultures were then incubated at 37°C in an orbital shaker at 220 rpm. Once the cultures reached the late exponential growth phase, we adjusted their OD600 to 1, as described above, and used one starter culture each to either inoculate the iron limited or the iron rich replicates. The experimental cultures consisted of 200 ml media in one-liter Erlenmeyer flasks, inoculated to an initial density of OD600 = 1x10-2. The growth of the PAO1 cultures was monitored with a spectrophotometer. The cultures were harvested for total RNA isolation at mid to late exponential phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cultures were transferred onto ice. We then distributed them to 50 ml tubes (Greiner) containing 5 ml ice cold stop solution (10% phenol buffered with Tris-HCl to pH8 (Sigma-Aldrich, Switzerland) and 90% ethanol). The suspension was mixed by manual inversion of tubes and centrifuged at 4°C, for 5 minutes at 9000 rpm. The supernatant was discarded, while the cell pellet was flash-frozen in liquid nitrogen and stored at -80°C. RNA was harvested using hot phenol protocol (Pessi et al 2007). Afterwards cDNA was produced. The supernatant treatments were each mixed with CAA and independently generated supernatants. So we used three biological repliactes of each supernatant to generate three independend biological replicates of our PAO1 culture for RNA isolation.
RNA libraries were prepared using Encore Complete Prokaryotic RNA-Seq DR Muliplex System
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
The sequences of the reads were quality trimmed using the program fastq_quality_trimmer
Sequencing reads were mapped to the P. aeruginosa PAO1 genome sequence (Stover et al. 2000) from the Pseudomonas Genome Database (Winsor et al. 2016) using the CLC Genomics Workbench v7.0 (QIAGEN CLC bio, Aarhus, DenmarkCLC bio) allowing up to 2 mismatches per read.
RNA-Seq count data were subsequently analyzed with the Bioconductor package DESeq2 version 1.6.3 (Love et al. 2014) in the R environment version 3.1.2 (R Development Core Team 2014).
Assembly: P. aeruginosa PAO1 genome sequence (Stover et al. 2000)
Supplementary files format and content: txt files include expression values, unique and total gene reads, RPKM and mean values for each sample
|
| |
|
| Submission date |
Feb 08, 2023 |
| Last update date |
Dec 31, 2023 |
| Contact name |
Gabriella Pessi |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Zürich
|
| Street address |
Winterthurerstrasse 190
|
| City |
Zürich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL18782 |
| Series (1) |
| GSE224821 |
RNAseq reveals that Pseudomonas aeruginosa mounts medium-dependent competitive responses when sensing diffusible cues from Burkholderia cenocepacia |
|
| Relations |
| BioSample |
SAMN33210719 |
| SRA |
SRX19313139 |
