Full-length cDNA-seq of Homo sapiens: ECSCs (endometrial carcinoma-derived stem-like cells), Replicate b
Cancer tissue samples used to isolate CSCs were obtained during surgical resection and were kindly provided by the Forschungsverbund BioMedizin Bielefeld/OWL (FBMB) at the Protestant Hospital of Bethel Foundation (Bielefeld, Germany) after assuring routine histopathological analysis. A primary tumor sample was collected from an endometrioid carcinoma. Informed consent according to local and international guidelines was signed by the patient and further experimental procedures were ethically approved (Ethics committee Mnster, Germany, 2017-522-f-S).  Samples were transferred to ice-cold Dulbeccos Phosphate Buffered Saline (PBS; Sigma Aldrich, Munich, Germany) and for further processing transported to the University of Bielefeld. Tumor tissue was mechanically disintegrated followed by enzymatic digestion with collagenase for 2 h at 37C. The minced tissue was cultured on gelatin-coated (bovine skin-derived, type B; Sigma Aldrich) culture dishes in CSC-selective medium composed of Dulbeccos Modified Eagles medium/Hams F-12 (Sigma Aldrich) with the addition of 2 mM L-Glutamin (Sigma Aldrich), Penicillin/Streptomycin (100g/mL) epidermal growth factor (20 ng/mL; MiltenyiBiotec, Bergisch Gladbach, Germany), fibroblast growth factor-2 (40 ng/mL; Miltenyi Biotec), B27 supplement (Gibco, Thermo Fisher Scientific, Bremen, Germany) and 10% FCS (Sigma Aldrich). RNA from 1x10^6 cultured CSCs of each population and cancer type were isolated by using the NucleoSpin RNA Plus kit (Macherey-Nagel, Dren, Germany) according to manufacturers guidelines. Quality and concentration of isolated RNAs were assessed via nanodrop ultraviolet spectrophotometry and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Bblingen, Germany). Total RNA samples with RNA Integrity Numbers (RIN) > 9.5 were used to convert full-length RNA molecules that are both capped and polyadenylated to cDNA using the TeloPrime Full-Length cDNA Amplification Kit V2 (Lexogen, Austria). Amplified full length cDNA were then used to prepare Oxford Nanopore Technologies (ONT) compatible libraries using the Ligation Sequencing Kit LSK109 with the Native Barcoding Kit NBD104 (ONT, UK), which were in turn run on three R9.4 flow cells on the ONT system GridION. Base calling and demultiplexing were performed using Guppy v3.1.5.