characteristics.text: 2025-04-30
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE)
characteristics.characteristic: INSDC center name
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS23999178
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-15035:Sample 1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: macrophage
characteristics.characteristic: cell type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Bacterial strains Yersinia enterocolitica strains used in this study are derivatives of the serotype O:8 strain WA314 harboring the virulence plasmid pYVO8. Several mutant strains (WA314ΔYopM, WA314ΔYopP, WA314ΔYopMP) were generated and used, as detailed in Table S1.  Cell culture Human peripheral blood monocytes were isolated from buffy coats as described in Kopp et al., 2006. Cells were cultured in RPMI1640 with 20% autologous serum at 37°C and 5% CO₂. Medium was changed every three days until differentiation into macrophages by day 7. Infections were typically performed one week after isolation, except for one batch (Table S2), where cells were used after 2 weeks.  Infection of cells The day before infection, the medium was changed to antibiotic- and serum-free RPMI1640. Y. enterocolitica strains were grown overnight in LB with antibiotics at 27°C. On the day of infection, cultures were diluted 1:20 in antibiotic-free LB, incubated for 90 min at 37°C to induce T3SS. Bacteria were pelleted, resuspended in PBS with 1 mM MgCl₂ and CaCl₂, and adjusted to OD600 = 3.6. Macrophages were infected at MOI 100, and centrifuged briefly to synchronize infection. RNA-seq Sample Preparation Total RNA was isolated from 1–2 x 10⁶ infected human macrophages using the RNeasy extraction kit (Qiagen) including on-column DNase treatment, following the manufacturer's instructions. RNA integrity was assessed using RNA 6000 Nano Chip on an Agilent 2100 Bioanalyzer. mRNA enrichment was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina, following the manufacturer’s instructions. Library concentration was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Fragment size distribution was analyzed on an Agilent 2100 Bioanalyzer using the DNA High Sensitivity Chip. Final libraries were normalized to 2 nM and pooled equimolar.
characteristics.characteristic: description
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: blood
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Y. enterocolitica mock
characteristics.characteristic: stimulus
characteristics.text: Sample 1
characteristics.characteristic: title