DNA sensing and Interferon Beta 1 transactivation drive radiation-induced anti-tumor immune effects in human pancreatic cancer cells
DNA sensing and Interferon Beta 1 transactivation drive radiation-induced anti-tumor immune effects in human pancreatic cancer cells
The pancreatic cancer cell lines PANC-1, MIA Paca-2 and BxPc-3 were purchased from ATCC. PANC-1 and MIA Paca-2 were in cultured in Dulbecco's Modified Eagle Medium (DMEM) medium, supplemented with 10% fetal bovine serum (FCS) and 1% penicillin/streptomycin (P/S). BxPC-3 were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium, supplemented with 10% FCS, 1% P/S and 1% L-Glutamine.  All cells were maintained at at 37 °C in a humidified atmosphere with 95% air and 5% CO2. Cells were grown to 70-80% of confluency at time or irradiation. Prior to irradiation, cell culture flasks were filled air-bubble free with unsupplemented medium, which was replaced immediately after irradiation with fresh, supplemented medium. RNA-seq samples were prepared in three biological replicates. Cells were harvested and counted, 20% Drosophila S2 cells were added as a spike-in control. Cell pellets were resuspended in 1 mL TRI reagent (Sigma). 200 µL Chloroform (Applichem) were added, samples were mixed and centrifuged at 4°C, max. speed for 15 min. The upper phase was transferred to a new tube and subjected to isopropanol precipitation. 20 µg of RNA were treated with 40 U DNase I (Roche) at 37°C for 30 min and purified by phenol-chloroform extraction and Ethanol precipitation. rRNA was depleted and RNA-seq libraries were prepared with NEBNext rRNA depletion kit v2 (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions.