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| Status |
Public on Sep 27, 2023 |
| Title |
PAO1_pit2_NI_1 |
| Sample type |
SRA |
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| Source name |
PAO1
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| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: PAO1
genotype: single genomic integration of pit2 via pUC18mini-Tn7-lac-GW
treatment: 1 mM IPTG
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| Treatment protocol |
Induced conditions: CAA was supplemented with 1 mM IPTG
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| Growth protocol |
LB overnight cultures of PAO1::empty and PAO1::pit2 in CAA + 150 µM FeCl3 were diluted a 100-fold in 50 mL CAA medium to an OD600nm of 2
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| Extracted molecule |
total RNA |
| Extraction protocol |
At OD (600 nm) = 2, 2 mL cells was harvested, 20 % ice cold stop mix solution (95 % v/v ethanol, 5 % v/v phenol, saturated pH 4.5) was added and the mixture was immediately snap-frozen in liquid nitrogen. Next, samples were thawed on ice and their pellets were collected (20 min, 4 °C, 4500 g) for resuspension in 300 µL of a 0.5 mg/mL lysozyme solution in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Subsequently, samples were treated via the hot phenol method, followed by ethanol precipitation to extract the RNA. Next, the samples were treated with DNase I and the RNA was further purified using ethanol purification.
RNA samples were converted into cDNA using the Illumina Stranded Total RNA Prep Ligation Ribo Zero Plus kit. The Bioanalyzer and the DNA high sensitivity kit (Aligent) were used to verify rRNA depletion and evaluate the average fragment length, aiming for fragments around 500 bp.
All samples (n = 3 per condition) were pooled together in equimolar amounts to obtain a 25 µL sample of 4 nM and the cDNA samples were paired-end sequenced via Illumina next-generation sequencing (Novaseq 6000)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
total RNA with depletion of rRNA
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| Data processing |
cDNA reads quality and trimming
The quality of the cDNA reads was analyzed using FastQC (0.11.8) (Andrews, 2010). Adapters and poor quality sequences were trimmed using Trimmomatic (v0.39) (Bolger et al., 2014).
Allignment
BWA-MEM aligner (v0.7.17) was used to map the read pairs to the reference genome of PAO1 (NC_002516.2), which was manually altered to carry the pit2 genomic insert in its Tn7 site.
Normalization
The resulting fragment counts were normalized by gene length and the total number of mapped non-rRNA fragments per sample to yield fragments per kilobase of transcript per million mapped fragments (FPKM) values
statistics
Principal Component Analysis (PCA) was conducted with the prcomp package in R to evaluate sample clustering. Finally, gene expression analysis was performed using the DESeq2 Bioconductor package in R (v1.30.0)
Assembly: PAO1 (NC_002516.2)
Supplementary files format and content: FeatureCounts results containing raw gene feature counts and counts normalized by fragments per kilobase per million mapped non-rRNA fragments
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| Submission date |
Mar 19, 2023 |
| Last update date |
Sep 27, 2023 |
| Contact name |
Rob Lavigne |
| E-mail(s) |
rob.lavigne@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Biosystems
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| Lab |
Laboratory of Gene Technology
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| Street address |
Kasteelpark Arenberg 21
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| City |
Leuven |
| ZIP/Postal code |
3001 |
| Country |
Belgium |
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| Platform ID |
GPL26913 |
| Series (1) |
| GSE227677 |
The phage-encoded protein PIT2 impacts bacterial quorum sensing in Pseudomonas aeruginosa by direct interaction with LasR |
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| Relations |
| BioSample |
SAMN33819915 |
| SRA |
SRX19720107 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7105076_PAO1_pit2_NI_1_counts.csv.gz |
60.6 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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