Analysis of epithelial and mesenchymal populations in tumor-derived cell lines by scRNA-seq
Analysis of epithelial and mesenchymal populations in tumor-derived cell lines by scRNA-seq
Tumor-explant-derived cell lines were collected from GEM model primary pancreatic tumors using mechanical and chemical dissociation with 2mg/mL Dispase and 2mg/mL Collagenase IV. After 1h incubation at 37C 5%CO2, cells were centrifuged at 200 g for 5 minutes. Single cell suspensions were plated in a sufficient amount of medium. Short-term cultures were obtained; epithelial (EPI) and mesenchymal (MS) populations from the same tumor were derived with ganciclovir (GCV) treatment (5uM) in vitro. After stabilization of EPI and MS populations, cells were collected with trypsinization, centrifuged at 200g for 5 minutes, resuspended in Bambanker and stored in liquid nitrogen for downstream processing Nucleic acids were directly processed on intact cells following standard Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics) using  Chromium platform (10x Genomics). Target cell recovery was 5'000. Libraries were prepared using Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics). Library quality was assessed using TapeStation (Agilent Technologies). Libraries were sequenced on NovaSeq 6000 platform (Illumina) aiming at 50'000 clusters/cell.