GSM8511648: HTEC cDNA sample 1-1; Homo sapiens; RNA-Seq
Human tracheal epithelial cells were harvested from deceased organ donors according to established protocols (PMID: 1616056). Frozen cell aliquots were reactivated and cultured in epithelial growth media (EGM) [3:1 (v/v) F-12 Nutrient Mixture (Gibco)–Dulbecco's modified Eagle's medium (Invitrogen), 5\% fetal bovine serum (Gibco), 0.4 ug/mL hydrocortisone (Sigma-Aldrich), 5 ug/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Invitrogen), 24 ug/mL adenine (Sigma-Aldrich), and 10 uM Y-27632 (Enzo Life Sciences)] on 10 mm dishes coated with rat tail collagen (Sigma-Aldrich). EGM was changed three times a week until dishes were confluent, at which point the cells were passaged with 0.25\% trypsin for 30 minutes. For air liquid interface culture, expanded basal cells were plated at 50,000 cells per 6.5 mm transwell insert (Corning 3470) coated with human placental collagen (Sigma-Aldrich) and cultured with Pneumacult ALI (StemCell) for 21-28 days according to the manufacturer's instructions. Starting on day 27, interferon stimulation (IFN-beta: 10 ng/ml, IFN-alpha2: 10 ng/ml, IFN-gamma: 10 ng/ml, IFN-lambda2: 10 ng/ml) was added at hours 0, 24, 39, 42, and 45 prior to harvesting (For final timepoints 3, 6, 9, 24, and 48 hours). On the day of harvest, basal media was aspirated and both basal and apical chambers were rinsed twice with PBS. Following two washes, trypsin-EDTA (0.25% Fisher cat. 25200072) was added to both the basal and apical chambers (300 ul basal, 100 ul apical) and incubated for 30 minutes at 37degree C while pipette mixing every 10 minutes. Trypsinization was quenched with 300 ul of maintenance media and transferred to a 1.5ml eppendorf tube (eppendorf cat. 022431021) and centrifuged at 350xg for 5 minutes at 4degree C. Cells were resuspended in 94 ul of cell staining buffer (Biolegend cat. 420201) and blocked with 5 ul of TruStain FcX (Biolegend cat. 422302) for 10 minutes on ice. Blocked cells were stained with 1 ul of Biolegend Totalseq-B hashtags (Biolegend Totalseq-B hashtags 1-11) for 30 minutes on ice. Staining was quenched with 1 ml of cell staining buffer and spun at 300xg for 5 minutes at 4degree C prior to two more washes with 1 ml of cell staining buffer. Cells were resuspended in 100 ul of 0.05% BSA in PBS and counted via Countess II (Fisher cat. A27977). Counted cells were pooled equally into two pools and spun at 300xg for 5 minutes at 4degree C.