characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-03-31T00:22:23Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-03-31T00:22:23Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112197399
characteristics.characteristic: External Id
characteristics.text: Departamento de Desarrollo Biotecnologico, Instituto de Higiene, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay
characteristics.characteristic: INSDC center name
characteristics.text: 2023-03-31T00:22:23Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-03-31T00:22:23Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14308472
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12444:AMA40 - IHC1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: Protocols: AMA40 strain (control, wild-type), AMA40 IHC1 (Intracolony 1 Strain), AMA40 IHC 2 (Intracolony 2 Strain) The wild-type strain was recovered from a Mini-BAL sample in the year 2017. The mutant strains (IHC) were obtained after AMA40 was exposed to 4% human pleural fluid. The IHC strains were randomly selected within the inhibition ellipse from the cefidercol MICs. All strains were properly store at -80 °C and open in CLED and LB agar when required to perform the following experiments. Acinetobacter baumannii AMA40 strain was growth in 4% human Pleural Fluid (Treatment condition) and LB medium (control condition). The incubation was performed during 24 hours at 37 C. The intracolonies were recovered within the inhibition ellipse from the cefidercol MICs performed in iron-depleted cation adjusted Muller-Hinton. Acinetobacter baumannii exposed to 4% Human Pleural Fluid during 24 hours at 37 C. As control condition, AMA40 was growth in LB medium during 24 hours at 37 C. Whole genomic DNA was extracted with the Wizard Promega (Promega, Madison, USA) according to manufacturer instructions. 1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube. 2. Centrifuge at 13,000-16,000 × g for 2 minutes to pellet the cells. Remove the supernatant. For Gram Positive Bacteria, proceed to Step 3. For Gram Negative Bacteria go directly to Step 6. 3. Add 600μl of Nuclei Lysis Solution. Gently pipet until the cells are resuspended. 4. Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature. 5. Add 3μl of RNase Solution to the cell lysate. Invert the tube 2-5 times to mix. 6. Incubate at 37°C for 15-60 minutes. Cool the sample to room temperature. 7. Add 200μl of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate. 8. Incubate the sample on ice for 5 minutes. 9. Centrifuge at 13,000-16,000 × g for 3 minutes. 10. the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube containing 600μl of room temperature isopropanol. Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein. 11. Gently mix by inversion until the thread-like strands of DNA form a visible mass. 12. Centrifuge at 13,000-16,000 × g for 2 minutes. 13. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600μl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet. 14. Centrifuge at 13,000-16,000 × g for 2 minutes. Carefully aspirate the ethanol. 15. Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-15 minutes. 16. Add 100μl of DNA Rehydration Solution to the tube and rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4°C. 17. Store the DNA at 2-8°C. Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10bp UDI indices.
characteristics.characteristic: description
characteristics.text: exposed to 4% human pleural fluid
characteristics.characteristic: environmental stress
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: LB agar
characteristics.characteristic: growth condition
characteristics.text: Acinetobacter baumannii
characteristics.characteristic: organism
characteristics.text: Acinetobacter baumannii
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: cefiderocol resistant
characteristics.characteristic: phenotype
characteristics.text: Acinetobacter baumannii
characteristics.characteristic: scientific_name
characteristics.text: AMA40
characteristics.characteristic: strain
characteristics.text: AMA40 - IHC1
characteristics.characteristic: title