title: Co-culture of fibroblasts and pancreatic ductal adenocarcinoma cells with mouse peripheral ganglia
design: Co-culture of fibroblasts and pancreatic ductal adenocarcinoma cells with mouse peripheral ganglia
library_construction_protocol: Ganglia cells were seeded as described above and after axons started to grow out, cells were incubated for 1 hour with cell-trace violet (ThermoFisher C34557) to enable labelling. Cells were washed several times, fresh medium was added and PACO10-GFP (Sigma Aldrich MISSION® pLKO.1-puro-CMV-TurboGFP™ Positive Control Plasmid DNA) cells were added to the well. The cells were cultured together for 3 days before FACS sorting of PACO cells. For cell proliferation, ganglia cells were seeded as described above and PACO/fibroblast were cultured in a transwell chamber. RNA extraction and purification of FACS-sorted cells was done using PicoPure RNA Isolation Kit according to manufacturer's instructions (Thermo Fisher, cat # KIT0214). RNA quality assessment and quantification were performed with Bioanalyzer using Agilent RNA 6000 Pico Kit (Agilent, cat # 5067-1513). Whole transcriptome amplification was performed using a modified smart-seq2 protocol [49], with 5μl of a modified RT buffer containing 1× SMART First Strand Buffer (Takara Bio Clontech, cat # 639538), 1 mM dithiothreitol (Takara Bio Clontech), 1 μM template switching oligo (IDT), 10 U μl−1 SMARTScribe (Takara Bio Clontech, cat # 639538) and 1 U μl−1 RNasin Plus RNase Inhibitor (Promega, cat # N2615). Tagmentation of cDNA was done using Nextera XT DNA Library Preparation Kit (Illumina, cat # FC-121-1030).