Fixed RNA Profiling of HPAEpiCs Cultured at the ALI
Fixed RNA Profiling of HPAEpiCs Cultured at the ALI
HPAEpiCs on ALI day 28 were washed with PBS, incubated in 0.25% trypsin-EDTA, detached from the inserts, and separated by pipetting. Trypsinization was stopped by adding Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum. The cell suspension was then centrifuged, washed, and passed through a 40 µm Flowmi cell strainer (Bel-Art Products, Wayne, NJ, USA). The obtained cells were fixed with 4% formaldehyde in PBS, and subsequently treated with the Quenching Buffer and Enhancer contained in a Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). Cell barcoding, amplification, and library construction were performed using a Chromium Fixed RNA Profiling Reagent Kit (10x Genomics) in accordance with the manufacturer's protocol. Isolated single cells were loaded onto a 10x Chromium X (10x Genomics), barcoded, and pre-amplified to construct the sequencing library.