organism: Homo sapiens
description: Protocols: HBECs were grown and differentiated at an air-liquid interface (ALI) forming an in vitro 3D-lung model. In short, HBECs were grown and differentiated on 6.5 mm semipermeable transwell inserts (0.4 µm Pore Polyester Membrane Insert, Corning) and after two weeks at air-liquid interface (ALI) the cultures reached full differentiation, which was assessed using light microscopy focusing on epithelial morphology, presence of ciliated cells, and mucus production along with immunofluorescence staining for ciliated cells (acetylated-tubulin, T6793, Sigma) and goblet cells (muc5AC, Ab-1 (45M1), #MS-145-P, ThermoFisher). Primary human bronchial epithelial cells (HBEC) were isolated from proximal airway tissue obtained with informed consent from a patient. At 96 h post infection the mock treated, infected and infected +betamethasone treated HBEC ALI-cultures were washed three times on both sides with PBS. The HBEC ALI-cultures were then lysed and RNA extraction was done using the NucleoSpin RNA II kit (Macherey-Nagel) following the manufacturer's instructions. RNA-seq libraries were prepared using the Smart-seq2 method SARS-CoV-2 infection: The clinical isolate SARS-CoV 2/01/human/2020/SWE (GeneBank accession no. MT093571.1) was kindly provided by the Public Health Agency of Sweden. Vero E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 5% FBS (HyClone), 100 U/mL penicillin and 100 µg/ml streptomycin (PeSt, HyClone) at 37°C in 5% CO2. Propagation of the virus was done once in Vero E6 cells for 72 h and titration was done by plaque assay. The apical side of the HBEC ALI-cultures were rinsed three times with warm PBS shortly before infection. 15000 plaque forming units (PFU) of SARS-CoV-2 was added to the apical compartment in a total volume of 100µl infection medium (DMEM/PeSt), corresponding to an approximate multiplicity of infection (MOI) of 0.05. The HBEC ALI cultures were incubated at 37°C and 5% CO2 for 2.5 h before the inoculum was removed and the cultures were washed with PBS to remove residual medium.
identifiers.namespace: BioSample
identifiers.id: SAMEA114458272
attributes.tag: INSDC center name
attributes.value: Laboratory of Molecular Infection Medicine Sweden (MIMS), Umeå university
attributes.tag: Submitter Id
attributes.value: E-MTAB-12368:N709_S508_S66
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: lung fibroblast
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: infect
attributes.value: SARS-CoV-2 (01/human/2020/SWE)
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: lung
attributes.tag: sample name
attributes.value: E-MTAB-12368:N709_S508_S66
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: umuwell
attributes.value: tube4