Illumina NovaSeq 6000 paired end sequencing; Transcriptomic comparison of Listeria monocytogenes and its derivative mutant strain with inactivated lmo0946 gene in exponential phase of growth
Single colony of L. monocytogenes EGD-e its derivative mutant strain with inactivated lmo0946 were inoculated in BHI (Oxoid) and grown for 18 hours at 37 ◦C with aeration. RNA protect R Cell Reagent (Qiagen) was added to the cultures' samples according to the manufacturer's instructions. Cells were harvested by centrifugation, snap-frozen in liquid nitrogen, and stored at - 80 °C untill further processing.  The pellets were re-suspended in 1 mL of TRIGIDITY (Applichem) and cells disrupted by the Minilys® homogenizer (Bertin instruments). Total RNA was subsequently isolated with Direct-zol RNA MiniPrep (ZYMO™) according to the manufacturer's instructions. To remove DNA contamination, total RNA was incubated with TURBO DNAse (Ambion) in the presence of SUPERase• In™ RNase Inhibitor (Ambion) followed by purification using TURBO DNA- free™ Kit (Ambion). Concentration and assessment of total RNA quality was performed on a NanoDrop and Agilent 2100 Bioanalyzer, respectively. Ribosomal RNA depletion, library preparation and sequencing were performed by Eurofins Genomics Europe Sequencing GmbH. To enrich mRNA and remove ribosomal RNA (rRNA) from total RNA, total RNA was treated with Bacterial rRNA Depletion Kit (NEB). Preparation of cDNA fragment libraries was performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (Illumina).