Analysis of epithelial and mesenchymal populations in tumor-derived cell lines by scGET-seq
Analysis of epithelial and mesenchymal populations in tumor-derived cell lines by scGET-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 permeabilized nuclei following the  “Nuclei Isolation for Single Cell ATAC Sequencing” protocol CG000169, Rev D (10X Genomics). Specifically, we prepared the nuclei suspension in order to get 5'000 nuclei as target nuclei recovery. Single-cell GET-seq was performed on Chromium platform (10x Genomics) following “Chromium Single Cell ATAC Reagent Kits” user manual version CG000168 Rev C. For GET-seq protocol, Tn5 and TnH were used in substitution of the 10X ATAC enzyme (10X Genomics) in the transposition mix assembly step. When a sequential Tn5 to TnH reaction was performed, a transposition mix containing 1.5 uL of Tn5 was incubated 30 min at 37 °C, then 1.5 uL of TnH was added and the reaction was continued for a total of 1 h incubation.