Kingfisher instrument

Total RNA was extracted using the MagMAX™ mirVana™ RNA Isolation Kit (Thermo Fischer Scientific, Waltham, MA, USA) on an automated KingFisher instrument (Thermo Fischer Scientific, Waltham, MA, USA). The MagMAX™ mirVana™ RNA Isolation Kit is a magnetic bead-based kit that uses MagMAX magnetic-bead technology for efficient isolation of total RNA from plasma samples from 100 µl of human plasma.

RNA extraction for diagnosis was performed using the KingFisher instrument (ThermoFisher Scientific, Waltham, MA, USA), starting from 300 µL of nasopharyngeal sample and obtaining 50 µL of eluted volume. For variant identification assays, 900 µL of nasopharyngeal sample was extracted in three 300 µL batches, using the EasyMag (Biomerieux, Marcy-l'Etoile, France) automated extraction system, and obtaining 150 µL of RNA.

RNA extraction from frozen nasopharyngeal swabs and processed mouse tissue samples was performed by using a custom Maxwell HT viral TNA (total nucleic acid) kit (Promega), optimized for a KingFisher instrument (Thermo Fisher), according to the manufacturer’s instructions.

We selected all cases with 2 sequential COVID-19 episodes at an interval of 20–45 days by considering the time between the last positive reverse transcription PCR (RT-PCR) specimen in the first episode and the first positive specimen in the second episode. We also requested cases for which >1 positive specimen was available in our stored collection, among those taken in the first 10 days of each sequential episode, and for which the specimens had sufficient viral load (cycle threshold [Ct] <32) to maximize the chance of obtaining optimal coverage in whole-genome sequence analysis. To minimize the possibility of including potentially persistent cases, we excluded cases that had clinical conditions or admissions to hospital services that likely corresponded to immunocompromised status.
We used remnants of nasopharyngeal swab specimens previously used for diagnostic purposes via TaqPath COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, https://www.thermofisher.com) during November 26, 2021–August 21, 2022. We extracted viral RNA from nasopharyngeal exudates by using the KingFisher instrument (ThermoFisher Scientific). We used 16 μL of RNA as a template for reverse transcription by using LunaScript RT SuperMix Kit (New England Biolabs, https://www.neb.com).

The 50 tissue samples from CRSwNP patients and healthy controls were directly put in RNA later medium (Thermo Fisher Scientific Inc., CA, USA) and processed for extraction and purification of total RNA using the Kingfisher RNA kit together with the Kingfisher instrument (Thermo Fisher Scientific Inc., CA, USA) according to the manufacturer’s instructions. The quantity and quality of isolated RNA was determined using the NanoDrop 2000 Spectrophotometer (Thermo Fischer Scientific) and 2200 TapeStation Automated Electrophoresis System (Agilent Technologies). Samples with an RNA integrity number of greater than 6.0 were chosen for sequencing, with the exception of one polyp sample at RIN 4 which was included.