|
| Status |
Public on Feb 07, 2024 |
| Title |
YERS053, Resistant, Time 60 minutes, Replicate A |
| Sample type |
SRA |
| |
|
| Source name |
culture
|
| Organism |
Yersinia pestis |
| Characteristics |
time: 60 minutes
strain: YERS053
resistance: Ciprofloxacin resistant
treatment: 0.25 ng/ul Ciprofloxacin
|
| Treatment protocol |
One milliliter of diluted cells were treated with indicated concentrations of ciprofloxacin and incubated at 37°C for 60 minutes (or indicated time) in 5 ml Eppendorf tubes (Thermo Fisher Scientific) with constant agitation.
|
| Growth protocol |
Glycerol stocks were plated on sheep’s blood agar plates, grown overnight at 37°C, and selected for recovery the following day in tryptic soy broth (TSB). Cells were recovered in TSB for approximately 3 hours then diluted to a McFarland standard of 0.5 using the DensiChek Plus
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2 volumes of RNAprotect Bacteria Reagent was added to each tube and incubated at room temperature for 5 minutes at indicated time points. For all reactions tubes were centrifuged at 5000 x g for 10 minutes. The supernatant was decanted, and pellets were extracted or stored at -80°C until processing. Pellets were resuspended in 100µl 1xTE containing 15 mg/ml lysozyme and 10 µl Proteinase K. Suspended pellets were incubated at room temperature for 10 minutes. Then, 700 µl of buffer RLT was added to suspended pellets along with approximately 100 µl of 0.5 mM glass beads and cells were incubated at 95°C for 5 minutes, bead beaten for 10 minutes, then centrifuged at max speed for 30 seconds. 760 µl of supernatant was added to new 2 ml Eppendorf tubes containing 590 µl of 80% ethanol. Samples were then extracted utilizing the RNeasy kit protocol and an on-column DNase treatment according to manufacturer’s protocols
Extracted RNA was quantified using the Qubit 2.0 Fluorometer and Qubit RNA HS assay. RNA was spiked with the ERCC control, an external RNA control, and input into the QIAseq FastSelect – 5s/16s/23s kit to remove bacterial ribosomal RNA prior to sequencing. Sequencing libraries were prepared using the QIAseq Stranded total RNA library kit according to manufacturer’s instructions. Libraries were quantified using the High Sensitivity D1000 kit on the 4200 Tapestation System. Four barcoded libraries were combined and diluted to a 4 nM library pool, denatured with NaOH, and further diluted to a final library concentration of 12 pM prior to sequencing. Pooled libraries were sequenced using the MiSeq Instrument and MiSeq reagent kit version 3–150 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
YERS053_R_60_A
|
| Data processing |
FASTQ files were imported into CLC Genomics workbench and analyzed using the RNA-Seq Analysis workflow. Reads were mapped using mapping setting: mismatch cost (2), insertion cost (2), deletion cost (3), length fraction (0.8), and similarity fraction (0.8). Paired reads were counted as two when calculating transcripts per million (TPM).
Assembly: GCF_000008445.1, GCF_000222975.1
Supplementary files format and content: Excel file containing raw TPM counts from CLC genomics workbench
|
| |
|
| Submission date |
Aug 22, 2023 |
| Last update date |
Feb 07, 2024 |
| Contact name |
Christopher Patrick Stefan |
| E-mail(s) |
christopher.p.stefan.civ@health.mil
|
| Organization name |
USAMRIID
|
| Street address |
1425 Porter Street
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL33703 |
| Series (1) |
| GSE241489 |
Relative quantification of the recA gene for antibiotic susceptibility testing in response to ciprofloxacin for pathogens of concern |
|
| Relations |
| BioSample |
SAMN37115143 |
| SRA |
SRX21456067 |
