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Sample GSM6600478 Query DataSets for GSM6600478
Status Public on Nov 04, 2022
Title Serum_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 and 20% human serum (Serum)
condition: Serum
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 SAMN31017273
SRA SRX17706408

Supplementary file Size Download File type/resource
GSM6600478_Serum_5.wig.gz 346.2 Kb (ftp)(http) WIG
GSM6600478_Serum_5_tradis_gene_insert_sites.csv.gz 140.2 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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