title: GSM4387449: ∆prfA rep 2; Listeria monocytogenes; RNA-Seq
library_construction_protocol: For each sample, 10 mL of RNAprotect bacteria reagent (Qiagen, Valencia, CA) was added to 10 mL of bacterial culture. The mixture was then incubated at room temperature for 10 min to ensure that the bacterial RNA was stabilized. Cells were pelleted by centrifugation (4,637 × g, 30 min) at 4°C and then lysed with 25 mg/ml proteinase K (Thermo Fisher Scientific, Waltham, MA) and 50 mg/ml lysozyme (Thermo Fisher Scientific) at 37°C for 30 min. Cell lysates were subsequently mixed with 1 mL of Tri Reagent (Ambion, Austin, TX), transferred into bead-beating tubes that contained 3 mL of 0.1 mm zirconium beads, followed by adjustment (with Tri Reagent) to a final volume of 5 mL. The lysates were processed in the bead-beater at maximum speed for 4 min. After bead-beating, samples were centrifuged (4,637 × g, 10 min) at 4°C. Supernatants were separated from the beads, transferred into a new tube, and mixed with 500 μL of bromo-chloro-propane (BCP). After incubation with BCP for 10 min, samples were centrifuged at 14,637 × g for 15 min at 4°C. The aqueous layer was collected, and nucleic acids were precipitated overnight at 80°C using 2.5 volumes of 100% ice-cold isopropanol. Nucleic acids were pelleted, washed with 75% ethanol, as resuspended in 100 μL nuclease free water. Total RNA was incubated with Turbo DNase (Life Technologies) to remove remaining DNA in the presence of RNasin (Promega). Subsequently, RNA was purified using phenol-chloroform/chloroform extractions, followed by precipitation and resuspension. Quality of RNA was assessed using a 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA) (all samples had RNA integrity numbers > 5). Efficiency of the DNase treatment was determined using qRT-PCR for the housekeeping gene rpoB (all samples showed Ct > 35, indicating absence of DNA contamination at relevant levels). All experiments were performed in biological triplicate. Preparation of directional cDNA fragment libraries was performed using the ScriptSeq Complete Kit (Bacteria) (Illumina, San Diego, CA). 16S and 23S rRNA was removed from total RNA with Ribo-Zero rRNA Removal Reagents (Bacteria) and Magnetic Core Kit. rRNA-depleted samples were run on the 2100 Bioanalyzer (Agilent Technology) to confirm reduction of 16S and 23S rRNA and followed by purification using Agencourt RNAClean XP Kit (Beckman Coulter Inc, Brea, CA). The Cornell Core Facility for RNA-sequencing prepared and sequenced indexed RNA-seq libraries (ScriptSeq Complete Kit, Illumina). Sequencing was carried out on a NextSeq 500 (single-end, 75 bp per read).
attributes.tag: GEO Accession
attributes.value: GSM4387449