title: WT_1904
organism: Homo sapiens
description: Protocols: hAEC cultures from 2 different human donors were inoculated in duplicate at the apical surface with 10,000 TCID50 of either the WT or NS1R38A virus or Hank's Balanced Salt Solution (HBSS) as mock (untreated) control and incubated for 1 hour at 37°C in a humidified incubator with 5% CO2. Afterwards inoculum was removed, and the apical surface was washed three times with HBSS, after which the cells were incubated for an additional 17 hours at 37°C in a humidified incubator with 5% CO2. The apical surface of the remaining inserts was washed four times with 200 µL of HBSS followed by a final washing step of both the apical and basolateral surface with 200 and 500 µL of HBSS, respectively. Cells were dissociated from the Transwell® insert by adding 200 and 500 µL of TrypLE (Thermo Fisher Scientific) to apical and basolateral compartment and an incubation step of 10 minutes at 37°C in a humidified incubator with 5% CO2. This was followed by a gentle disruption of the cell layer through pipetting using a large bore-size pipette tip, and an additional incubation of 20 minutes at 37°C in a humidified incubator with 5% CO2. Dissociated cells were transferred into 800 µL wash solution, which is composed of Air-Liquid Interface (ALI) medium supplemented with 0.1% Pluronic (Thermo Fisher Scientific), and the remaining clumps were gently disrupted through pipetting using a large bore-size pipette tip. Next three cycles of centrifugation for 5 minutes at 250x rcf and resuspension in 1 mL washing solution were performed. Afterward the cells were resuspended in 300 µL washing solution and the cell number, cell viability and cell size were assessed with trypan blue on a Countess II (Thermo Fisher Scientific). Isolation and culturing of primary material was performed as previously described (Jonsdottir J. & Dijkman, R. Characterization of human coronaviruses on well-differentiated human airway epithelial cell cultures. in Coronaviruses: Methods and Protocols vol. 1282 73-87 (Springer New York, 2015). The hAEC cultures were allowed to differentiate for at least six weeks at 37°C in a humidified incubator with 5% CO2 to ascertain the visual appearance of a well-differentiated pseudostratified layer with ciliary beating and mucus production prior to use. The single cell partitioning was performed on a Chromium Controller (10x Genomics) using the Chromium Single Cell 3' Reagent Kit (version 2, 10x Genomics) according to manufacture protocol. The obtained partitions were further processed using the Chromium Single Cell 3' Reagent Kit (version 2, 10x Genomics) according to manufacture protocol, to generate Nextera XT sequencing libraries.