Differential analysis of Escherichia coli transcriptomes following a short overproduction of the OmrA or OmrB small RNA, wt or mutated in their 5'-end conserved region.
The effect of OmrA or OmrB, wt or mutated in their 5'-end conserved region, was analyzed on the Escherichia coli K-12 transcriptome by RNAseq. For this, cells deleted of the chromosomal omrAB locus, and transformed by plasmids carrying the desired omr gene under the control of a PLlac0-1 inducible promoter, were grown to mid-exponential phase. The Omr sRNAs were then induced by addition of 100 mM IPTG for 10 minutes, and total RNA was extracted with hot phenol. After DNase treatment, 3 mg of total RNA were depleted of rRNA using the RiboZero bacterial kit (Epicentre) and used for libraries preparation with the TruSeq Stranded RNA kit (Illumina). After Qubit quantification, the libraries were sequenced on a HiSeq 2500 Illumina sequencing machine (single read, 50 bases).