NextSeq 550 paired end sequencing; Bulk RNA-seq of an ALI (Air-lung interface) model of SARS-CoV2
Bulk RNA-seq of an ALI (Air-lung interface) model of SARS-CoV2
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. 72 h.p.i the cells were washed three times with PBS and RNA was harvested from mock-infected (n=1) and infected (n=2) HBEC from individual donors using the NucleoSpin RNA II kit (Macherey-Nagel) in accordance with the manufacturer's instructions. According to smartseq2, https://www.nature.com/articles/nprot.2014.006