Transcriptomic analysis reveals autophagy flux variation within mouse pancreatic cancer organoids

Murine pancreatic cancer cells (HY15549 cells) established from genetically engineered mouse models (GEMM) of PDAC (p48-Cre+, KrasLSL-G12D/+, Trp53lox/+; KPC mice) were transfected with the GFP-LC3-RFP autophagy flux reporter from Mizushima lab, wherein reduction in the GFP/RFP ratio indicates increase in autophagy flux. Organoids derived from these cells were dissociated into single cells and sorted into autophagy-high (AThi) and autophagy-low (ATlo) populations according to GFP/RFP signal ratio. To validate this reporter, RNA was extracted from these two populations and expression of autophagy/lysosome related genes was examined. Overall design: RNA was isolated from sorted AThi and ATlo populations (n = 2 and 3 biological replicates), which were used for RNA-sequencing to obtain mRNA expression profiles of these two populations.