RNA-seq EHEC M9 control rep1
RNA-Seq expression profiling: RNA isolationA transcriptomic assay was performed using the RNA-seq methodology to obtain a gene expression profile of EHEC O157:H7 EDL933 strain phagocytized by RAW 264.7 murine macrophages or under stress conditions at 2 h post-incubation. Control RNA was isolated from bacterial cells grown in DMEM and M9 minimal medium. RNA was obtained using the Monarch Total RNA Miniprep Kit (New England Biolabs, USA). The sample was enriched with bacterial RNA using the MICROBEnrich kit (Ambion, USA) to eliminate eukaryotic RNA, which employs magnetic beads coated with oligonucleotides that hybridize to the polyadenylated 3 ends of eukaryotic mRNAs. RNA integrity was verified using an Agilent Bioanalyzer 2100 and concentration was measured using the Qubit fluorometer.Library preparation and sequencingTotal RNA was treated with RiboZero (Illumina Inc., USA) to remove ribosomal RNA (rRNA). For samples obtained from the RAW 264.7 infection, rRNA removal was performed using the RiboZero Plus protocol (Illumina Inc., USA). Samples from controls and EHEC exposed to multi-stress in vitro were processed with the RiboZero Bacterial protocol (Illumina Inc., USA). Libraries were quantified with Qubit (Thermo Fisher Scientific, USA) and analyzed with the Fragment Bioanalyzer (Agilent, USA). Single-end cDNA libraries for 300 bp fragments were constructed following the Illumina TruSeq protocol (Illumina Inc., USA). cDNAs were purified by beads and analyzed by capillary electrophoresis in the Agilent Bioanalyzer 2100. Sequencing was performed on the Illumina MiSeq platform (Illumina Inc., USA), generating approximately 70 million reads for phagocytized EHEC samples and 10 million reads for EHEC subjected to stress conditions.