Illumina MiSeq paired end sequencing; Transcriptional response to NusG depletion in Salmonella
Transcriptional response to NusG depletion in Salmonella
Upon reaching the OD600 value specified in the \"treatment protocols\", bacterial cultures (4 ml) were centrifuged for 2 min at 14,000 rcf. Pellets were quickly resuspended in 0.6 ml of ice-cold REB Buffer, no SDS (20 mM sodium acetate pH 5.0, 10% sucrose) and processed for RNA extraction.  Bacterial cultures were inoculated from single colonies and grown overnight in LB medium at the 37°C. Overnight cultures were treated as specified in protocols 2 through 5. The culture was diluted 1:200 in LB medium and grown with shaking at 37°C to an 0D600 of 0.5. Bacterial suspensions were mixed with one volume of phenol (Aquaphenol TM from MP biomedicals saturated with water and preheated to 65°C). The mixture was shaken vigorously for 5 min at room temperature. The aqueous phase was recovered after 2 min of centrifugation at 14,000 rcf and extracted for 5 min at room temperature with one volume of a 1:1 phenol chloroform mixture. The mixture was centrifuged for 2 min at 14,000 rcf and extraction was repeated with one volume of chloroform. The aqueous phase was mixed with 55 µl of 3M Sodium Acetate  (pH 5.0) followed by the addition of 2.5 volume of 95% Ethanol. RNA was precipitated overnight at -20°C. The pellet was recovered by centrifugation (14,000 rcf for 15 min at 4°C), dried under vacuum and resuspended in 12 µl of water. Library construction RNA was quality-controlled on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies). 2 µg of total RNA were treated with DNAse (Baseline Zero DNAse, Epicentre) prior to ribosomal RNA depletion using the RiboZero Bacteria magnetic Kit from Illumina, according to the manufacturer recommendations. Samples were then checked on the Agilent Bioanalyzer for proper rRNA depletion. Directional RNA-seq libraries were constructed using ScriptSeq V2 RNA-Seq library preparation kit (Illumina), according to the manufacturer recommendations.
Experimental Factor: compound
none
Experimental Factor: phenotype
wild type