|
| Status |
Public on Mar 09, 2022 |
| Title |
14_day_rep_1 |
| Sample type |
SRA |
| |
|
| Source name |
bacterial cells, pelleted
|
| Organism |
Acinetobacter baumannii |
| Characteristics |
strain: ATCC 17978
genotype: WT
treatment: 14-day desiccation
|
| Treatment protocol |
A volume of 100 mL (containing 3x1010 CFU) per sample was spread sterilely across the surface of a 100 mm polystyrene petri dish. Petri dishes were incubated at ambient humidity and temperature. After 2 and 14 days of desiccation, bacteria were rehydrated in 1 mL of PBS. For 2-day samples, 5 ml of rehydrated bacteria were diluted into 10 mL of LB. For 14-day samples, 50 ml of rehydrated bacteria were diluted into 10 mL of LB. Cultures were grown for 4 hours at 37°C with shaking at 180 rpm to enrich for surviving transposon mutants. In parallel, input samples were prepared by inoculating transposon library aliquots into 10 mL LB medium and growing for 8 hours at 37°C with shaking at 180 rpm. Following outgrowth, bacterial cultures were pelleted and stored at -80°C.
|
| Growth protocol |
Transposon library aliquots were inoculated into 10 mL LB medium (three biological replicates per condition) and grown for 8 hours at 37°C with shaking at 180 rpm. Bacteria were then harvested, washed twice in PBS, and resuspended in PBS at a concentration of 3x1011 CFU/mL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was extracted from bacterial pellets using the Qiagen DNeasy Blood and Tissue kit according to manufacturer’s instructions.
gDNA was sheared by sonication using the Covaris LE220 instrument to generate 350 bp 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.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
library strategy: Tn-seq
Reads were trimmed
Reads were filtered for quality
Reads were mapped to the ATCC A. baumannii 17978 accession NZ_CP012004.
Using TUCF genomics analysis pipelines, a “Dval” score was assigned to each gene in each library pool, representing 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 for the library pool.
Output Dval scores were normalized to input Dval scores to calculate a fitness score for each gene in each respective condition.
Fitness scores were Log2-transformed and an average Log2 fitness score was calculated for each gene in each condition analyzed (2 and 14 days of desiccation).
A Z-score was calculated that represents the number of standard deviations from the mean for each respective gene
Genome_build: Acinetobacter baumannii ATCC 17978 NZ_CP012004
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 |
Mar 06, 2022 |
| Last update date |
Mar 15, 2022 |
| Contact name |
Erin R Green |
| E-mail(s) |
[email protected]
|
| Phone |
7243961121
|
| Organization name |
Vanderbilt University Medical Center
|
| Department |
Pathology, Microbiology, and Immunology
|
| Lab |
Skaar Lab
|
| Street address |
1161 21st Ave South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37212 |
| Country |
USA |
| |
|
| Platform ID |
GPL24655 |
| Series (1) |
| GSE198004 |
Tn-seq of desiccated Acinetobacter baumannii |
|
| Relations |
| BioSample |
SAMN26491162 |
| SRA |
SRX14390455 |
