GSM7867718: hBEC, signaling perturbations, donor no. 429581 [ali12]; Homo sapiens; RNA-Seq
HBECs were collected 14 days post-injury by treating with 0.05% Trypsin-EDTA (prepared by dilution of 0.25% Trypsin- EDTA, ThermoFisher Cat. # 25200056, with calcium/magnesium-free PBS), for 20 minutes at 37 °C. Trypsin dissociation was halted with Trypsin Neutralizer Solution (ThermoFisher Cat. # R002100) at 1:1 dilution and cells were filtered through a 40 μm strainer and pelleted by centrifugation at 300x g for 5 minutes. Cells were resuspended in phosphate buffered saline (ThermoFisher Cat. # 10010072) with 0.1% bovine serum albumin (ThermoFisher Cat. # B14) and counted on a Countess Cell Counter (ThermoFisher) to determine cell number and viability. For cell counting, 10 μL of cell suspension was mixed with 10 μL of Trypan Blue Solution (ThermoFisher Cat. # 15250061) and 10 μL of that 1:1 mix was loaded into Countess chip and read with default program. Viable cell numbers were used to determine cell concentration. Optiprep Density Gradient Medium (Sigma Cat. # D1556) was added to achieve a final concentration of 15% Optiprep and 120-150,000 cells/ml. Cells were captured by inDrops as detailed in Zilionis, R. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat. Protoc. 12, 44–73 (2017). inDrops v3 scRNA-seq; Library construction as detailed in Zilionis, R. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat. Protoc. 12, 44–73 (2017).