title: Sample 2
organism: Homo sapiens
description: 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.
attributes.tag: ENA first public
attributes.value: 2025-04-30
attributes.tag: INSDC center name
attributes.value: Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE)
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15035:Sample 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: macrophage
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-15035:Sample 2
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: stimulus
attributes.value: Y. enterocolitica WA314