Regulation of Listeria monocytogenes PrfA regulon by environmental stimuli in gastrointestinal Tract
Bacterial cells were harvested by centrifugation at 13000 x g for 3 mins. Listeria monocytogenes were grown in MD10 media consisting of 49 mM K2HPO4, 11 mM NaH2PO4, 1.7 mM MgSO4, 187 µM ferric ammonium citrate, 9.3 mM NH4Cl, 49.5 mM glucose or glycerol, 2.96 µM thiamine‐HCl, 1.33 µM riboflavin, 2.05 µM biotin, 24 µM lipoic acid, 0.1 g l−1l‐cysteine and l‐leucine, 0.2 g l−1l‐arginine, l‐histidine, l‐methionine, l‐valine and l‐isoleucine. Additionally, the medium was supplemented with 0.1 % w/v case‐amino acids. For microaerobic growth (oxygen 5 %-6 % v/v, carbon dioxide 10 % v/v and nitrogen 85 % v/v) L. monocytogenes were grown in a VAIN incubator (Du scientific) in 6 well plates (Coster®) shaking at 140 rpm respectively at 37 C. Carbon source was glucose or glycerol. RNA extraction was carried out using Invitrogen's Purelink RNA Extraction Kit and Lysing Matrix E tubes (MP Biomedicals). Prior to starting the extraction, fresh lysozyme solution (consisting of 10 mg/ml lysozyme, 10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA,) and a solution of 10% SDS (w/v) were prepared in RNAse-free water and the kit lysis buffer was supplemented with 10 μl of 2-mercaptoethanol per 1 ml of lysis buffer. Bacterial cells were harvested by centrifugation at 13000 x g for 3 mins. The supernatant was discarded and 100 μl of lysozyme solution was added to the pellet for cell resuspension. Next, 0.5 μl of SDS solution (10% v/v) and 350 μl of lysis buffer was added and vortexed sequentially. The lysate, added to a Lysing Matrix E tube, was homogenised in a rotor stator homogeniser (FastPrep FP120, 6.5 speed) for 45 secs. The tube was centrifuged for 5 mins at 2600 x g followed by transfer of the supernatant to a microcentrifuge tube (RNAse-free) and addition of 250 μl of absolute ethanol (100% v/v) to the tube. The solution mixture was vortexed to remove precipitate. The sample was then transferred to a spin cartridge and centrifuged at 12000 x g for 15 secs allowing RNA to bind to the spin column. The subsequent flow-through was discarded and 700 μl of Wash Buffer 1 was added to the column. This was then centrifuged again for 15 secs at 12000 x g, followed by discarding both the flow through and the collection tube. The column was then placed in a new collection tube. Wash Buffer 2 (500 μl) was added to the column and centrifuged at 12000 x g for 15 secs, again discarding the subsequent flow-through. The wash step was repeated with Wash Buffer 2 and flow-through was discarded again. The column was centrifuged at 12000 x g to remove any residual wash buffer. The collection tube was discarded and the column was transferred to a RNAse-free recovery tube; 50 μl of RNAse-free water was carefully added to the column membrane. RNA was eluted in this 1.5 ml tube with 1 min incubation at room followed by centrifugation at 12000 x g for 2 mins. All further experiments involving RNA were performed on ice. The Nanodrop spectrophotometer (ND-1000) was used for RNA quantification using 1 μl of the sample. Additionally, the Super RNAse Inhibitor (Invitrogen) was added to the samples at 1U/μl to inhibit RNAse activity. Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. In addition, Illumina ran Listeria monocytogenes fasta files through their in house in-silico analysis program which predicted we would need some additional probes to efficiently deplete the rRNA from this species. These were purchased from from Integrated DNA Technologies, Inc.(https://eu.idtdna.com/) and added during the probe hybridization step.