GSM9193206: F. tularensis LVS treated with vehicle control, replicate 5; Francisella tularensis; RNA-Seq
Broth cultures of F. tularensis LVS treated with vehicle, dillapiole (8 mg/ml), or no treatment were grown to stationary phase and normalized to the lowest OD600 and pelleted. After removing the supernatant, the pellets were resuspended in Trizol and incubated at room temperature for 5 minutes. Chloroform was then added and vigorously mixed for 15 seconds until the sample turned milky pink. The mixture was then transferred to an organic matrix tube and centrifuged at max speed for 15 minutes. After centrifugation, the aqueous phase was transferred to a new tube and 70% ethanol was added at a 1:1 ratio. The resulting mixture was then transferred to a spin column (Invitrogen PureLinkTM RNA Mini Kit) and spun at max speed for 15 seconds. After the flow through was discarded, PureLink Dnase (Invitrogen) was added directly to the column and incubated for 15 minutes are room temperature. Following Dnase treatment, Wash Buffer 1 was added to the columns and centrifuged for 15 seconds. The columns were then washed with Wash Buffer 2 twice; for each wash, the columns were centrifuged for 15 seconds followed by a final 2 minute spin to ensure the column was dry. Spin columns were transferred to recovery tubes and Rnase free water was added to elute RNA. The columns incubated at room temperature for 2 minutes before centrifuging for 1 minute. The RNA concentrations were determined using a NanoDrop spectrophotometer and then stored at -80°C For RNA-Seq analysis of Francisella cells (hexane vs dillapiole treated), total RNA was extracted from eight replicate samples in each experimental group. RNA Integrity Number (RIN) values as determined by Bioanalyzer electrophoresis were all above 9. RNA samples (2 mg) were dissolved in 15 ml TE buffer (10mM Tris HCL pH 8.0, 1mM EDTA). 16S and 23S ribosomal RNA (rRNA) were removed using the MICROBExpress Bacterial mRNA Enrichment kit (Life Technologies). rRNA-depleted RNA samples were then resuspended in Rnase-free water and quantitated by fluorescence-based Qubit RNA HS Assay kit (ThermoFisher Scientific) and fragmented for 4 minutes at 94ºC to be used as input for RNA library preparation. Illumina-compatible RNA libraries were prepared using KAPA RNA HyperPrep Kit (KAPA Biosystems, Inc). Library quality and insert size were assessed by electrophoresis on Agilent DNA High Sensitivity DNA chips; average library size was ~ 370 bp. Libraries were quantitated using fluorescence-based Qubit dsDNA HS Assay (ThermoFisher Scientific). Purified libraries were clustered and sequenced on an Illumina HiSeq1500 in a 2 x50 paired end Rapid Run at the Marshall University Genomics Core Facility.