Single cell RNA sequencing analysis of alveolar epithelial type II cells (AE2) isolated from young and old mice with and without lung injury

The objective of the analysis was to investiage how AE2 cell function is effected by age and lung injury at single cell transcriptomic level Overall design: For single cell RNA sequencing analysis, young and old mice treated with saline or LPS were sacrificed 24 h later. The lungs were digested and a cell suspension was generated. For the current experiment, a cell hashing based experimental approach was used to demultiplex pooled cell samples. This approach uses oligo-tagged antibodies to label cells prior to mixing. Accordingly, the harvested lung cell suspension was incubated with biotinylated EpCAM antibody (BioLegend #118203) for 30 min on ice, followed by washing steps and 30 min incubation with oligo barcoded PE streptavidin TotalSeq™-A antibodies (BioLegend #405251, 405253, 405255, 405257) as described in the supplier's manual. For each experimental group (young control, young 24 h LPS, old control, old 24 h LPS) a different oligo barcoded antibody was used. The cells were then sorted for EpCAM positive cells. DAPI was used to detect and exclude dead cells. The harvested cells were counted and the four experimental groups were pooled in equal number for scRNA-seq analysis. The pooled samples were loaded on one 10x Genomics lane. The whole experiment was performed twice in the form of an independent biological replica experiment.