|
| Status |
Public on Apr 12, 2021 |
| Title |
PAO1161 EV_3_control_for_PA3458_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
empty vector control (EV)
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: PAO1161/pKGB8 (araC araBADp)
genotype: empty vector control (EV)
growth phase: logarithmic phase of liquid culture
|
| Treatment protocol |
All cultures were incubated at 37oC in L broth (DifcoTM L Broth, Lennox, BD) supplemented with 75 μg/mL chloramphenicol and 0.02% arabinose with shaking until reaching exponential phase of growth (OD600 = 0.5).
|
| Growth protocol |
The competent P. aeruginosa PAO1161 cells were prepared as described previously (Irani and Rowe, 1997) and transformed using pKKB2.11 carrying araC araBADp-PA3458 or empty vector (araC araBADp) to obtain PAO1161/ pKKB2.11 (araC araBADp-PA3458 ) and PAO1161/ pKGB8 (araC araBADp), respectively. Transformants were selected on L agar plates supplemented with 150 μg/ml chloramphenicol and were verified by isolation of plasmid DNA and its digestion. Bacteria from single colonies were used to inoculate L broth (DifcoTM L Broth, Lennox, BD) liquid cultures with supplementation (75 µg/ml chloramphenicol) and grown overnight with shaking at 37oC. Three independent overnight cultures for each strain were diluted 1:100 into fresh L broth with chloramphenicol (75 mg/ml) and 0.02% arabinose and grown with shaking at 37oC. Samples from the cultures were taken at regular intervals to measure the optical density at 600 nm (OD600). Aliquots of 2 ml from logarithmic phase cultures (OD600 = 0.5) were subjected to RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from three independent replicates of each strain. 2 ml of cultures which reached optical density 0.4-0.6 at 600 nm were mixed with 4 ml of RNAprotect Bacteria Reagent (Qiagen). RNA was isolated with an RNeasy mini-kit (Qiagen) according to the manufacturer’s protocol. Total RNA was digested with DNase (TURBO DNA-free Kit, Ambion) to eliminate genomic DNA.
rRNA was depleted using Ribo-Zero rRNA Removal Kit (Bacteria) (MRZMB126,Illumina) according to manufacturer instructions. Libraries were prepared according to instructions accompanying the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, E7370S).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
gene expression data from empty vector control
PAO1161 EV_3
|
| Data processing |
Sequencing data were quality-checked and filtered using FASTP version 0.20.0.
Reads were mapped to P. aeruginosa PAO11161 genome (CP032126) using Bowtie2 version 2.3.4.3 using default settings.
The number of reads mapping to individual genes was counted using FeatureCounts v 2.0.1 (with the -s2 option).
Differential expression analysis was conducted using edgeR ver 3.28.0.
Genome_build: CP032126
Supplementary_files_format_and_content: Raw counts of sequencing (FeatureCounts output summarized for all samples).
A table with DE data for all genes and normalized abundance measurements (EdgeR output).
|
| |
|
| Submission date |
Feb 20, 2021 |
| Last update date |
Oct 28, 2021 |
| Contact name |
Adam Kawałek |
| E-mail(s) |
[email protected]
|
| Phone |
+48225921216
|
| Organization name |
Institute of Biochemistry and Biophysics, PAS
|
| Department |
Department of Microbial Biochemistry
|
| Street address |
Pawinskiego 5A
|
| City |
Warszawa |
| ZIP/Postal code |
02-106 |
| Country |
Poland |
| |
|
| Platform ID |
GPL21297 |
| Series (2) |
| GSE163235 |
Defining the regulons of Pseudomonas aeruginosa transcriptional regulators |
| GSE167147 |
MarR type regulator PA3458 is involved in osmoadaptation control in Pseudomonas aeruginosa (RNA-seq) |
|
| Relations |
| Alternative to |
GSM5660407 |
| BioSample |
SAMN18015763 |
| SRA |
SRX10142062 |
