RNA-Seq of Xanthomonas phaseoli pv. manihotis: a double recombinant mutant lacking rpfC, rpfG and rpfH and transformed with the empty vector pBBR1-MCS5
Total RNA was extracted using a modified hot phenol-chloroform manual extraction method. Briefly, each strain was grown on solid medium (NYGA) and a single colony was subcultured in 30 ml of NYG medium and incubated in constant agitation for 18 hours from an initial OD600 of 0.002; cells were harvested at 18 hours. Cells were then centrifuged, placed on ice, and mixed with hot acid phenol (pH 4.5) and a lysis buffer solution consisting of 4M LiCl, SDS 2%, 0.5M EDTA (pH 8) and 1M Tris-HCl (pH 8). Cells were then vortexed and a solution of chloroform:isoamyl alcohol (24:1) was added prior to a second vortexing and centrifugation step to separate the different phases. Two overnight precipitation steps were carried out: the first with 4M lithium chloride and the second one with 3M sodium acetate (pH 5.2). RNA quality was confirmed using a bioanalyzer (Agilent 2100, Santa Clara, CA). Two independent extraction processes were performed with different colonies for each strain to account for two biological replicates. Subsequent procedures were performed at the Beijing Genome Institute (BGI, Hong Kong): rRNA depletion was performed with the Ribo-ZeroTM rRNA Removal Kit from Epicentre (Madison, Wisconsin); RNA-Seq was performed through the Illumina HiSeq 2000 platform. Libraries generated were paired-end (100bp read length) and strand-specific.