title: Toward biomarker-guided radiotherapy: Multi-omics analysis links ATM status to radioresistance in lung adenocarcinoma
design: Toward biomarker-guided radiotherapy: Multi-omics analysis links ATM status to radioresistance in lung adenocarcinoma
library_construction_protocol: Flasks were washed with 2*5ml ice-cold PBS, then cells were scraped with 2 ml ice-cold PBS and were sedemented by centrifugation (500xg, 15 min, 4 °C). NA Cells were seeded 2 days before experiemtna and were grown in RPMI with 10 % FCS supplemented with Glutamax, Penicilin and Streptomycin at 37 °C and 5 % CO2 in humidified atmosphere. Samples were transported in a thermo-insulated container and irradiated with 6 MV photons at a dose of 2 Gy using a Clinac 2300 C/D linear accelerator (Varian). Non-irradiated controls were transported in parallel under identical conditions.  Sequence images were transformed with BaseCaller Illumina software to BCL files and demultiplexed to fastq files with bcl2fastq v2.20.0.422. Sequencing quality was determined using FastQC v. 0.11.5 software Following 15 min incubation, 200 µl chloroform were added, and the mixture was gently inverted to mix phase. After centrifugation (12,000 rpm, 15 min, 4 °C), the aqueous phase was transferred to a fresh reaction tube, and RNA was precipitated by addition of 500 µl isopropanol. After centrifugation under the same conditions, the supernatant was discarded, and the pellet was air-dried. RNA was resuspended in RNase-free water and quantified spectrophotometrically. RNA quality was assessed by measuring the RNA integrity number (RIN) using a Fragment Analyzer HS Total RNA Kit (Advanced Analytical Technologies, Inc.). Library preparation for RNA-Seq was performed in the STAR Hamilton NGS automation using the Illumina Stranded mRNA Prep (Cat.N° 20040534) and the ID for Illumina RNA UD Indexes Set A, Ligation with 96 Indexes (Cat. N° 20091646) starting from 300 ng of total RNA. The size range of the final cDNA libraries was determined by applying the SS NGS Fragment 1- to 6000-bp Kit on the Fragment Analyzer (average 340 bp). Accurate quantification of cDNA libraries was performed by using the DeNovix DS-Series System.