Illumina HiSeq 2000 paired end sequencing; RNA-seq analysis of Pseudomonas syringae pv. tomato DC3000
RNA-seq analysis of Pseudomonas syringae pv. tomato DC3000
The strains were grown on LB plates for 48 hours, resuspended in 10 mM MgCl2 and adjusted to an OD660 of 2.0. Two μl aliquots were inoculated in the centre of of PAG plates (0.5% protease peptone No. 3 (Difco 212693) and 0.2% glucose with 0.5% Difco Bacto-Agar) and incubated at 25°C for 24 hours (wild type) or 25 hours (mutant). Swarmer cells were resuspended in 5 ml MgCl2 10 mM and harvested, washed with sarkosyl 0.1% and cell pellets were immediately frozen in liquid nitrogen and conserved at -80oC until RNA isolation. rRNA removal with Ribo zero magnetic kit was performed by Macrogen. The cells were processed for RNA isolation using TRI Reagent®LS (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer instructions, except that the TRI Reagent was preheated at 70oC and the lysis step was carried out at 65oC. The RNA obtained was treated with RNase-free DNase I (Quiagen) followed by purification with RNeasy mini kit (Quiagen).RNA concentration was determined spectrophotometrically and its integrity was assessed by agarose gel electrophoresis. cDNA libraries were constructed by Macrogen.