nasal transcriptomics: Sample G125922-01
RNA was isolated from cells swabbed from the inferior nasal turbinate using the KingFisher Flex sample purification system (ThermoFisher) and the quick DNA-RNA MagBead kit (Zymo Research) following the manufacturer's instructions. Ribosomal depletion, cDNA synthesis, and library construction steps were performed using the Total Stranded RNA Prep with Ribo-Zero Plus kit following the manufacturer's instructions (Illumina). All steps were automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch-to-batch variability and increase sample throughput. Final cDNA libraries were quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution was checked using a fragment analyzer (Advanced Analytical; kit ID DNF474). Samples where adapter dimers constituted more than 4% of the electropherogram area, were failed prior to sequencing. Successful libraries were normalized to 10nM for sequencing.  Barcoded libraries were pooled. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on the NovaSeq 6000 sequencer using S4 flowcells with a target depth of 50 million 100 base-pair paired-end reads per sample (25 million read pairs).