|
| Status |
Public on May 14, 2024 |
| Title |
Input library (untreated) |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cell
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: Newman
cell type: Bacterial cell
treatment: Transposon library before treatment
|
| Treatment protocol |
The three different treatment conditions included: vehicle control (60:40 calprotectin buffer to TSB ratio), 300 µg/mL of WTCP, or 600 µg/mL of ∆s1CP.
|
| Growth protocol |
A single aliquot of frozen S. aureus Newman Transposon library was inoculated 1:50 in 5 mL of TSB and grown for 1 hour at 37°C with shaking at 180 rpm. An aliquot of this input library was also saved for sequencing. After 1 hour of growth, the library was subcultured 1:100 into 5.5 mL aliquots of three different treatment conditions. At 6 hours, cultures were pelleted and stored at -80°C. This experiment was performed in triplicate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA (gDNA) from S. aureus cultures was extracted using the DNeasy Blood and Tissue Kit (QIAGEN). All DNA samples were then normalized to 100 ng/mL in ultra-pure water. Samples were then processed using the DTR gel filtration cartridges (Edge Biosystems Inc 42453) using the manufacturer’s recommendations.
The sample (50 mL) was sheared in Covaris tubes using the Covaris LE220 instrument to generate 350bp fragments. Sheared DNA was treated with terminal deoxytransferase to generate a 3’ poly C-tail sequence, and two rounds of nested PCR were employed to amplify transposon junction regions. These products were multiplexed using 8-bp indexing primers and sequenced on the Illumina Hi-Seq 2500 at Tufts University Core Facility.
Transposon sequencing
|
| |
|
| Library strategy |
Tn-Seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Following sequencing, raw files were uploaded to the Galaxy Server from Tufts University to trim the reads, filter for quality, map to the S. aureus genome, and determine fitness using a “Dval” value for every gene in each library pool. A “Dval” score represents the aggregate number of reads for all transposon insertions within a gene in a given library sample, divided by the total number of predicted reads for that gene based on its size and the total number of reads obtained on the library pool. Dval scores in calprotectin treatments were normalized to Dval scores in vehicle control to calculate a fitness score for each gene in each experimental condition. Fitness scores were Log2 transformed and used to calculate a Z-score using the average and standard deviation of the population. A Z-score cutoff for significance was set at two standard deviations away from the mean.
Assembly: Staphylococcus aureus genome (NC_009641)
Supplementary files format and content: File is in .xlsx format. Contains raw sequencing reads as well as normalized abundance (Dval) values and fitness analysis
|
| |
|
| Submission date |
Apr 26, 2024 |
| Last update date |
May 14, 2024 |
| Contact name |
Eric P Skaar |
| Organization name |
Vanderbilt University Medical Center
|
| Department |
Pathology, Microbiology, and Immunology
|
| Lab |
Eric Skaar
|
| Street address |
1161 21st Avenue South
|
| City |
Nashville |
| State/province |
Tennessee |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL19006 |
| Series (1) |
| GSE266003 |
Tn-seq of Staphylococcus aureus exposed to Calprotectin |
|
| Relations |
| BioSample |
SAMN41097502 |
| SRA |
SRX24381215 |
