RNA sequencing of MDA-MB-231 cell line: total RNA
The MDA-MB-231 human breast cancer cells were grown in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% FBS, 2mM L-Glutamine, a mixture of 100 U/ml Penicillin/100 g/ml Streptomycin and with the addition of 1% non-essential amino acids. The cells were maintained in a humidified atmosphere at 37C and 5% CO2. The cells were maintained in a humidified atmosphere at 37C and 5% CO2. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Milan, Italy), checked for quality and purity the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) selecting RNA extracts with a RIN (RNA integrity number) value > 8. Two biological replicates for each RNA sample were sequenced. Total RNA of cells irradiated, using an electron accelerator, with the doses of 9 and 15 Gy delivered in Conventional or FLASH modality were also sequenced. Libraries were prepared using the CORALL RNA-Seq V2 Library Prep Kit (Lexogen, Vienna, Austria). Ribo-depleted RNAs serve as templates for CORALL reverse transcription, and no prior RNA fragmentation is required. Reactions were performed following the manufacturers instructions. Purification steps were based on the use of the Ampure XP magnetic beads (Beckman Coulter, Milan, Italy). After quantification with Qubit (Thermo Fisher Scientific, Milan, Italy), and quality control using the LabChip GX (PerkinElmer, Waltham, MA, USA), the indexed libraries were pooled, quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland), and sequenced on the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). For each sample, raw sequencing data were pre-processed to extract the 10-base pair UMI sequence from each read and append it to the read header for subsequent duplicate identification. Following UMI extraction, Cutadapt v4.1 was employed to remove adapter sequences and trim low-quality bases of the reads to ensure high-quality data for alignment. The pre-processed reads were aligned to the human reference genome (assembly version GRCh38) using the aligner STAR v2.7.10b. After alignment, PCR duplicates were identified and removed using UMI-tools v1.1.4, which collapses reads with the same alignment coordinates and identical UMI into a single count, thus mitigating amplification bias. The resulting de-duplicated alignment files were stored and indexed in BAM format. Gene-level expression was quantified by counting the number of reads mapping to each gene annotated in the reference genome. This was performed using HTseq v2.0.2.