title: RNA-seq of four glioblastoma (GBM) cell lines treated with irradiation, PARP7i, diABZI alone or in combination with irradiation
design: RNA-seq of four glioblastoma (GBM) cell lines treated with irradiation, PARP7i, diABZI alone or in combination with irradiation
library_name: s_GB1_NT_3x8Gy_r3.SE_s
library_construction_protocol: GBM cells were mixed with 10% mouse embryonic fibroblasts (MEFs) as a spike-in control prior to RNA isolation. Cells were washed 1x in PBS, detached using trypsin-EDTA solution (Sigma) and pelleted by centrifugation at 500g for 5min.  The Human GBM cell lines U251 and T98G cell lines were 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). GB1 and KALS1 cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 21875-034), supplemented with 10 % FBS, 1% P/S and 1 % LGlutamine (Gibco, 25030-024). All cell lines were maintained at 37 °C in a humidified atmosphere with 95% air and 5% CO2. Cells were grown to 70-80% of confluency at time of irradiation. Prior to irradiation, cell culture flasks were filled air-bubble free with unsupplemented medium to accommodate the vertical flask positioning in both irradiation workflows, which was replaced immediately after irradiation with fresh, supplemented medium. X-ray irradiations were administered using a horizontal irradiation cabinet (YXLON, TU32-D03, 20 mA, 5.5FOC, filtration: 3 mm Be + 3 mm Al + 0.5 mm Cu). Dedicated irradiation set-ups for all flask types used (Nunc™ Lab-Tek™ ChamberFlasks (170920), T25 (169900) and T75 (156800; all Sarstedt) were developed to accommodate the horizontal beam geometry and ensure standardized positioning. Cells were irradiated with 8 Gy. Fractionation regimen of 3x8 Gy was implemented for the X-ray experiments. PARP7 inhibitor KMR-206 was used in concentration of 300 nM, and the STING agonist diABZI was used in concentration of 100nM.  Cell pellets were resuspended in 1 mL TRAzol (Neo-biotech). 200 µL chloroform (Applichem) was added and the lysate was vortexed and centrifuged at maximum speed (21100xg) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.5 mL isopropanol. The RNA pellet was isolated by centrifugation for 30 min at 4°C, washed with 1 mL 75% ethanol, re-centrifuged for 10 min, dried and resuspended in 70 µL RNase free water. 20 µg RNA was treated with 40 U DNaseI (Roche) for 30 min at 37°C and subsequently purified by phenol-chloroform extraction and ethanol precipitation. RNA-seq libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions using 600 ng total RNA as input.