Illumina HiSeq 2000 paired end sequencing; RNAseq of Pseudomonas extremaustralis under oxidative stress at low oxygen conditions
RNAseq of Pseudomonas extremaustralis under oxidative stress at low oxygen conditions
P. extremaustralis 14-3b was used trough the experiments. All cultures were grown in Lysogeny Broth (LB) at 30ºC. Cultures were incubated under aerobic conditions in Erlenmeyer flasks using 1:10 medium-to-flask volume ratio and high agitation (200 rpm) or under microaerobic conditions in sealed bottles using a 1:2 medium-to-flask volume ratio and low agitation (50 rpm). Microaerobic culture's medium was supplemented with KNO3 (0.08%). For oxidative stress experiments the cultures were aerobically or microaerobically grown for 24 h as described and then incubated for 1 h with 3 mM H2O2 (a-OS and m-OS conditions). Briefly, P. extremaustralis cultures grown under microaerobic or m-OS conditions were harvested and RNA was isolated using the Trizol method. RNA quality was analyzed on the Agilent Bioanalyzer and rRNA depletion was performed using the MICROB Express Kit (Ambion) with the addition of 5S oligos. Samples were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies), and the final concentration was measured using a Qubit 2.0 Fluorometer (Invitrogen).  Directional libraries were prepared with ScriptSeq v2RNA-Seq Library Preparation Kit (Epicentre) and were sequenced with the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 100 nt. For each condition duplicated independent RNA extraction and libraries were used.
Experimental Factor: stimulus
control sample-microaerobiosis