GSM7508303: CRSwNP_NP_1, Nasal polyps, scRNAseq; Homo sapiens; RNA-Seq
Each sample was received directly from surgeons and promptly delivered to the laboratory on ice. Upon arrival at the laboratory, the samples were immediately processed. The ethmoid sinus mucosa was removed from the bone and nasal polyps were left intact and were minced into small pieces by scissors on ice. The minced tissues were placed into a C tube (Miltenyi Biotec, Bergisch Gladbach, Germany) within a solution of RPMI 1640 (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS), 0.02 mg/ml DNase I (Millipore Sigma, St. Louis, MO), and 4 mg/ml collagenase type IV (Thermo Fisher Scientific). The mixture was homogenized using the gentleMACS Dissociator (Miltenyi Biotec) and incubated at 37°C for total of 30 minutes (15 minutes, 2 times) with rotated using MACSmix Tube Rotator (Miltenyi Biotec). Between and after the two incubations, they were also homogenized in a gentleMACS Dissociator. Finally, the samples were filtered through a 70-μm cell strainer and spun down at 500g for 5 min. Red blood cells (RBC) were lysed using the RBC Lysis Solution (BioLegend, San Diego, CA) for 4 min at room temperature. Cells were then washed with ice-cold PBS and spun down at 500g for 5 min at 4°C before resuspension in RPMI containing 10% FBS. The cell suspension was filtered through 40-μm Flowmi Cell Strainers (Millipore Sigma) and counted to adjust concentration to 1,000 cells per μl. In all samples, the process up to this point was completed within 3 hours to minimize cell dissociation artifacts. The single cell suspension was loaded onto the Chromium Controller (10x Genomics) using the Chromium single cell 3' Reagent Kit v3 (10X Genomics), and scRNA-seq libraries generated in accordance with the manufacturer's protocols.