PROS1 secreted by airway basal cells upon viral infection and interferon response modulates mild COVID-19
PROS1 secreted by airway basal cells upon viral infection and interferon response modulates mild COVID-19
On the days of the extraction, the cells were washed with D-PBS, which was collected in individual tubes. Then the cells were lifted using Triple Express (Gibco, 12604-013) for 15 minutes at 37oC. The cell suspension was then transferred into the respective tubes, and the transwells were washed once with D-PBS to collect all the cells. The dead cells were then removed using the EasySep™ Dead Cell Removal (Annexin V) Kit (StemCell Technologies, 17899) as described in manufacturer's protocol, with the only difference that the cells were collected in 2 mL polypropylene tubes (Sarstedt, 72693) and live cells were negatively separated using DynaMag2 magnet (Life technologies, 12321D), in order to avoid pouring of suspensions while working with the virus.  The cells were then tagged in FACS buffer using the BD Human multiplex tags (BD, 633781). After the incubation, the cells were washed three times, pooled together, and loaded onto the scRNA-seq BD Rhapsody Cartridge of single-cell RNA extraction, using the BD Rhapsody Cartridge Reagent Kit (no. 633731) according to the manufacturer's protocol.   The cells were then tagged in FACS buffer using the BD Human multiplex tags (BD, 633781). After the incubation, the cells were washed three times, pooled together, and loaded onto the scRNA-seq BD Rhapsody Cartridge of single-cell RNA extraction, using the BD Rhapsody Cartridge Reagent Kit (no. 633731) according to the manufacturer's protocol.   cDNA was synthesised on mRNA captured on the beads using BD Rhapsody cDNA Kit (Cat. No. 633773), following manufacture's protocol. Libraries of 399 genes (CVR EXP1) were prepared using BD Rhapsody Targeted mRNA and the Tag Amplification Kit (no. 633774) and primers from the BD Rhapsody Immune response Panel Hs (399 genes, BD 633750), as per manufacture's protocol.  Whole transcriptome libraries (CVR EXP5) were prepared using the Whole transcriptome Analysis and Sample Tag library Preparation kit (BD 633801), as per manufacture's protocol (2019 version).