Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils
Description
Purified blood neutrophils were transmigrated in vitro as previously described (Dobosh et al, STAR Methods) using ALI/ARDS mBAL supernatant obtained by mechanical dissociation on ice using an 18G needle and syringe, followed by differential centrifugation at 800g and 3,000g to obtain the cell- and bacteria-free supernatant. A2 neutrophils were transmigrated, as described above, into ALI/ARDS mBAL supplemented with either 10𝜇g/mL of IgG control (Biolegend) or 10𝜇g/mL of Anifrolumab (anti-IFNAR1, Creative biolabs). Treatment with IgG control antibody or Anifrolumab was continued at the same concentration for the first hour of incubation for SARS-CoV-2. RNA from non infected neutrophils was isolated using the Nucleospin RNA kit (Takara). RNA isolated from in vitro samples was sequenced on the Illumina NextSeq500 at 75bp paired-end with a target of 20 million reads per sample. FASTQ files were checked for quality and raw sequencing data was aligned to the human reference genome (GRCh38) using STAR (Version 2.5.2) and quantmode was used to generate raw transcript counts.