title: GSM8683905: primary tumor cell line from KC mice, with mPSC and macrophages, control-3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using the Monarch Total RNA Miniprep Kit in accordance with the manufacturer's protocol. RNA sequencing was performed at the Core Unit Bioinformatics, Medical Faculty, Ulm University.Ex vivo microtumor models were constructed using organoids, mouse primary pancreatic stellate cells (PSCs), and bone marrow-derived macrophages embedded in a matrix consisting of 75% Matrigel, 25% Type I collagen, 5 μg/mL fibronectin, and 2.5 μg/mL vitronectin. Total RNA was extracted by adding TRIzol reagent (Thermo Fisher, USA) to the gel after removing the supernatant and dissolving the gel by thorough mixing. Chloroform (Thermo Fisher, USA) was then added, followed by vigorous vortexing for 15 seconds. After incubation at room temperature for 3 minutes, the mixture was centrifuged at 12,000×g for 15 minutes at 4°C. The upper aqueous phase was carefully transferred to a fresh tube, mixed with an equal volume of ≥95% ethanol, and purified using the Monarch Total RNA Miniprep Kit (New England Biolabs, Germany) according to the manufacturer's protocol. 200ng of total RNA, measured by Qubit® analysis, was used as input material for library construction using TruSeq RNA Sample Preparation Kit v2 (Illumina). Correct size distribution of the libraries was determined on a DNA1000 ScreenTape® using a Tape Station 4200 (Agilent)