RNA-seq of two Pancreatic ductal adenocarcinoma (PDAC) cell lines BxPC-3 and PANC-1 treated with 3x8 Gy X-rays without or with the PARP7 inhibitors RBN-2397 and KMR-206
RNA-seq of two Pancreatic ductal adenocarcinoma (PDAC) cell lines BxPC-3 and PANC-1 treated with 3x8 Gy X-rays without or with the PARP7 inhibitors RBN-2397 and KMR-206
Cells were harvested and counted, PDAC cells were mixed with 10% mouse embryonic fibroblasts (MEFs) as a spike-in control.  ANC-1 was cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco, 41965-039), supplemented with 10% fetal bovine serum (FBS) (Sigma, F7524-500ml) and 1% penicillin/streptomycin (P/S) (Gibco, 15140-122). BxPC-3 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 21875-034), supplemented with 10% FBS, 1% P/S and 1% L-Glutamine (Gibco, 25030-024). ll cell lines were cultured at 37 °C in a humidified environment containing 95% air and 5% CO₂. ells reached 70–80% confluency at the time of irradiation. Before irradiation, culture flasks were filled without air bubbles using unsupplemented medium to allow vertical positioning during both irradiation workflows. Immediately after irradiation, this medium was replaced with fresh, supplemented medium. X-ray irradiations were carried out using a horizontal irradiation cabinet (YXLON, TU32-D03, 20 mA, 5.5 FOC, filtration: 3 mm Be + 3 mm Al + 0.5 mm Cu), while all carbon-ion (C-ion) irradiations were performed at MedAustron using the particle synchrotron with a horizontal experimental beamline. For the C-ion studies, custom-made cell holders were designed and dosimetrically validated to allow cell irradiation in water, in line with recommendations from a recent National Cancer Institute special panel. The positioning uncertainty was estimated at 0.3 mm. For both experimental configurations (ChamberFlask and T25 flasks), a spread-out Bragg peak (SOBP) with a width of 4 cm and a range at the 80% dose level (R80) of 10.6 cm, corresponding to a C-ion energy of 285 MeV/u, was established. The dose-averaged linear energy transfer (LETD) was calculated using GATE/Geant4-based Monte Carlo simulations that accounted for the beam nozzle of the MedAustron experimental beamline, including contributions from all primary and secondary particles. The scoring volume was defined as a cylindrical geometry with a diameter of approximately 80 mm. At a depth of 8 cm, corresponding to the cell position at the center of the SOBP, the LETD was determined to be 58 keV/μm. Cells were exposed to a physical dose of 8 Gy using either 200 kV X-rays or C-ions. In addition, a fractionated regimen of 3x8 Gy was applied.  PARP7 inhibitor RNB-2397 (1 μM; TargetMol Chemicals), the PARP7 inhibitor KMR-206 (300 nM) were added 24 h prior to irradiation and kept until harvesting 72 h after the last irradiation. Cell pellets were resuspended in 1 mL TRAzol reagent (Neo-biotech). 200 µL chloroform (Applichem) was added, samples were mixed and centrifuged at 4°C maximum 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, 50-100-3290) at 37°C for 30 min and purified by phenol-chloroform extraction and ethanol precipitation. Polyadenylated RNA was enriched and RNA-seq libraries were prepared with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs E7490) and NEBNext UltraExpress RNA Library Prep Kit for Illumina (New England Biolabs E3330) according to the manufacturer's instructions using 250 ng total RNA input.