Label-free Single Cell Phenotyping to Determine Tumor Cell Heterogeneity in Pancreatic Cancer in Real-time

Two mouse cell lines were used for the single cell RNA-seq experiment, an epithelial line (ID53631) and a mesenchymal (ID9091) either as controls or after treatment for 72 hours with FFX. Cells were trypsinised and counted after performing a dead cell removal (dead cell removal kit, Miltenyi Biotec). 30,000 single cells were loaded on a 10x Chromium Next GEM Chip G to create GEMs. For the GEM preparation, the barcoding and library preparation the 10x Chromium Single Cell 3’ v3.1. chemistry was used according to the manufacturers instructions (10x Genomics). Quality and size analysis of cDNAs and libraries was performed on an Agilent Bioanalyzer 2100 using HS DNA Kit (Agilent). Libraries were sequenced in a NovaseqX+ 10B-100 flowcell (Illumina) following the standard protocol. ScRNAseq alignment was performed using 10x Genomics Cell Ranger v7.1.0 with mm10 as reference genome. Starting from the cellranger output, the analysis was then performed using the SCANPY toolkit.