Single-cell RNA sequencing

Polyp and inferior turbinate samples from two patients with CRSwNP were rinsed three times with DMEM containing penicillin (100 U/ml) (MilliporeSigma, Burlington, MA), streptomycin (100 μg/ml) (MilliporeSigma), and amphotericin B (250 ng/ml) (MilliporeSigma). Single-cell dissociation was performed by incubating 1- to 2-cm samples in Liberase disruption solution (MilliporeSigma). Digestion was stopped with 5 ml of DMEM and 10% fetal bovine serum, and the digest contents were filtered through a 40-μm cell strainer. The filtrate was centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 5 ml of chilled 0.04% BSA in PBS and counted with a manual cytometer using trypan blue (Thermo Fisher Scientific, Waltham, MA). Chilled 0.04% BSA in PBS was added to achieve a target cell concentration of 700 to 1200 cells per μl. Cell suspensions were loaded on a GemCode Single-Cell instrument (10x Genomics, Pleasanton, CA) along with reverse transcription reagents, gel beads containing barcoded oligonucleotides, and oil to generate single-cell gel beads in emulsion. Using the GemCode Single-Cell 3′ Gel Bead and Library kit (10x Genomics), an scRNA-seq library was prepared. These sequencing libraries were loaded on an Illumina NextSeq 500 (Illumina, San Diego, CA) and demultiplexing, barcode processing, and single-cell 3′ counting were performed using the Cell Ranger Single-Cell Software Suite (10x Genomics).