To isolate pancreases, first the omentum was removed then the pancreas collected with careful exclusion of draining lymph nodes. The pancreas was then minced. Enzymatic digestion was used with 0.02% Pronase (Roche cat 10165921001), 0.05% Collagenase P (Roche, cat. 11249002001), and 0.1% DNAse (Roche, cat. 10104159001) in Gey's balanced salt solution (Sigma cat G9779) for 50 min. Digested tissue was then filtered through a 100-μm nylon mesh. Cells were centrifuged at 1300rpm for 5min, washed and then resuspended in GBSS containing 0.3% bovine serum albumin (Sigma Aldrich cat A2153). The cell suspension was centrifuged, decanted and resuspended into 8ml 28.7% (wt/vol) solution of Nycodenz (Sigma, no longer available. Also used 16.7% Optiprep[Sigma-Aldrich D1556]) overlayed with 6ml .3% BSA GBSS, then centrifuged at 1400g with no break for 20 min. The cells of interest separated into a fuzzy band just above the interface of the Nycodenz cushion and the GBSS. This band was harvested, and the cells were washed and resuspended in MACs buffer.  Samples were stained and sorted for CD45-, TER119-, CD24a-, CD31-, 7AAD-, Calcein Violet+, PDPN+. Sorted single-cell suspensions were converted to barcoded scRNA-seq libraries by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics), loading an estimated 6,000 cells per library and following the manufacturer's instructions. Samples were processed using kits pertaining to either the V2 barcoding chemistry of 10x Genomics. Single samples were processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel. For each replicate, all samples (non-malignant and tumor) were processed in parallel in the same thermal cycler. The final libraries were profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies) and quantified using the Kapa Library Quantification Kit (Kapa Biosystems).
Experimental Factor: disease
normal
Experimental Factor: sampling site
normal tissue