description: Protocols: Primary human bronchial epithelial cells (HBEC) were isolated with informed consent from lung tissue from of patients who underwent thoracic surgery. HBEC were grown in Bronchial Epithelial Cell Medium (BEpiCM, SC3211-b, SienCell) with recommended supplements (SC3262, SienCell) and 100 U PeSt. For differentiation 0.75 x105 cells were seeded onto a 6.5 mm semipermeable transwell insert (0.4 µm Pore Polyester Membrane Insert, Corning) in differentiation media (DMEM:BEpiCM 1:1 supplemented with 52 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 0.5 ng/ml human recombinant epidermal growth factor, 0.5 μg/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 50 nM retinoic acid (all from Sigma Aldrich) and PeSt according to (doi: 10.1152/ajplung.00311.2001. PubMed PMID: 11792627). Cells were maintained submerged for the first 7 days, after which the media was removed from the apical side and cells were grown at air-liquid interface for an additional two weeks to reach full differentiation. Media was replaced three times per week with addition of fresh retinoic acid to the media shortly before usage. Immediately prior to infection the apical side of the HBEC ALI cultures was rinsed three times with PBS. For infection, 1.5x104 PFU of SARS-CoV-2 in a total volume of 100 µl infection medium (DMEM + 100 U PeSt) was added to the apical compartment, corresponding to an approximate MOI of 0.05. Cells were incubated at 37 °C and 5 % CO2 for 2.5 h before the inoculum was removed and the cells washed with PBS to remove residual medium. According to the 10x single-cell protocol. Uninfected samples we handled according to the standard 10x protocol while infected samples underwent the methanol fixation protocol CG000136_Demonstrated_Protocol_MethanolFixationCells_RevE.pdf as provided by 10x genomics. 10x v3.1 3'rnaseq 8b (index T), already used for demultiplexing R1 is 16 bp CB, 12 bp UMI and R2 is the cDNA; 94b
attributes.tag: ENA first public
attributes.value: 2022-06-16
attributes.tag: ENA last update
attributes.value: 2022-06-16
attributes.tag: External Id
attributes.value: SAMEA14516085
attributes.tag: INSDC center alias
attributes.value: Laboratory of Molecular Infection Medicine Sweden (MIMS), Umea university
attributes.tag: INSDC center name
attributes.value: Laboratory of Molecular Infection Medicine Sweden (MIMS), Umea university
attributes.tag: INSDC first public
attributes.value: 2022-06-16T00:12:55Z
attributes.tag: INSDC last update
attributes.value: 2022-06-16T00:12:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10697_2:P17601_1006
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: bronchial epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: lung
attributes.tag: sample name
attributes.value: E-MTAB-10697_2:P17601_1006
attributes.tag: sex
attributes.value: mixed