|
| Status |
Public on Jan 19, 2021 |
| Title |
PAO1 pAlpA_3 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells grown to OD ∼0.3 to 0.5
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: wild-type
plasmid: pAlpA
|
| Treatment protocol |
IPTG was added (final concentration 1 mM) and cells were grown for another 90 min to mid-log-phase (i.e. an OD600 of ∼0.3 to 0.5)
|
| Growth protocol |
Overnight cultures were back-diluted to an OD600 of 0.01 in 200 mL LB with 30 µg/mL gentamicin and grown at 37°C with shaking for 2 hrs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Tri Reagent
Ribosomal RNAs were depleted using the Ribo-Zero Magnetic Kit (Bacteria) (Epicentre). Remaining RNA was used to generate libraries using the NEB-Next Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs). Libraries were sized-selected by PAGE, isolating fragments corresponding to 150–300 bp.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were trimmed for adaptor sequences and mapped to the LVS genome using bowtie2 v. 2.3.5.1 with parameters -N 1 -L 10 -i S,1,1.00 -D 20
Read counds were assigned to the genes and other genomic features using custom scripts using HTSeq-count
Differential expression analysis was performed with DESeq2
For visualization, the bam alignment files were sorted using samtools (version 1.3.1).
The sorted reads were split by strand and convereted to the bedgraph file format using bedtools (version 2.27.1).
Read depth was normalized to the total number of mapping fragments in sample 1A using custom R scripts.
The normalized bedgraph files were converted to the bigwig file format using the bedgraphToBigWig executable (downloaded from the USCS database) for visualization in a genome brower.
Genome_build: NC_002516.2
Supplementary_files_format_and_content: bgwig files were generated using the bedgraphtobigwig script package from UCSC; Scores represent normalized RNASeq read density
|
| |
|
| Submission date |
Jun 15, 2020 |
| Last update date |
Jan 20, 2021 |
| Contact name |
Simon L Dove |
| E-mail(s) |
[email protected]
|
| Organization name |
Boston Children's Hospital and Harvard Medical School
|
| Department |
Division of Infectious Diseases
|
| Lab |
Dove Laboratory
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL18782 |
| Series (2) |
| GSE152484 |
Control of a programmed cell death pathway in Pseudomonas aeruginosa by a ppGpp-responsive antiterminator [RNA-Seq] |
| GSE152485 |
Control of a programmed cell death pathway in Pseudomonas aeruginosa by a ppGpp-responsive antiterminator |
|
| Relations |
| BioSample |
SAMN15238213 |
| SRA |
SRX8549734 |
