|
| Status |
Public on Nov 04, 2022 |
| Title |
RPMI_5 |
| Sample type |
SRA |
| |
|
| Source name |
P. aeruginosa transposon insertion mutant cell pool
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
strain: H103
cell type: P. aeruginosa transposon insertion mutant cell pool
growth condition: RPMI-1640 supplemented with 5% MHB (RPMI)
condition: RPMI
|
| Growth protocol |
A TnSeq pool was constructed in Pseudomonas aeruginosa PAO1 strain H103 using the mariner Himar1 transposon vector pBT20 until at least 200,000 mutants were collected. Total genomic DNA was extracted from TnSeq pool at baseline prior to growth under various testing conditions. Tested growth conditions included LB (planktonic), Mueller-Hinton Broth (MHB), RPMI supplemented with MHB (RPMI), RPMI supplemented with MHB and human serum (Serum), in-vivo murine skin abscess model that was left untreated (Abs_unt), and air-liquid interface human N/TERT skin organoid model (skin).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the Dneasy Blood and Tissue kit (Qiagen). Concentration and purity was measured using a NanoDrop 2000 (ThermoFisher).
Four replicate PCR reactions were used to amplify the transposon-genome junctions and were pooled together. Two-sided size selection was performed with Agencourt AMPure XP magnetic beads. Illumina Nextera Index Kit v2 SetA was used for indexing.
|
| |
|
| Library strategy |
Tn-Seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Read quality was assessed using FastQC v0.11.6
Read alignment 1: BioTraDIS was run using default parameters to align reads using SMALT and determine insertion counts per gene.
Read alignment 2: Transit was run using TPP pre-processing tool to align reads using BWA V0.7.17 with default parameters to tabulate counts of reads mapped per TA site. The read counts were passed through the Gumbel method to calculate the probability of essentiality for each gene.
Any genes predicted to be essential by BioTraDIS or Gumbel in at least four of five replicates for experimental conditions or two of three replicates for the baseline T0 pool were compiled into a final list of "essential" genes.
Assembly: Pseudomonas aeruginosa PAO1 downloaded from Pseudomonas Genome database v17.1 (accession GCF_000006765.1; PseudoCap version 138)
Supplementary files format and content: BioTradis (CSV): Comma-delimited files containing a list of all PAO1 genes, along with statistics of the number of sequencing reads mapped to a given gene by BioTradis (eg. "read count", "insertion count", etc).
Supplementary files format and content: Transit (WIG): Number of reads aligned at each TA site in the PAO1 genome, as calculated by Transit.
|
| |
|
| Submission date |
Sep 26, 2022 |
| Last update date |
Nov 06, 2022 |
| Contact name |
Robert E. W. Hancock |
| E-mail(s) |
[email protected]
|
| Phone |
604 822 2682
|
| Organization name |
University of British Columbia
|
| Department |
Department of Microbiology & Immunology
|
| Lab |
The Hancock Lab
|
| Street address |
2259 - Lower Mall Research Station
|
| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V6T 1Z4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18782 |
| Series (1) |
| GSE214167 |
Surviving the host: microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions |
|
| Relations |
| BioSample |
SAMN31017278 |
| SRA |
SRX17706403 |
