Quantitative Analysis of OxyS regulatory Transcriptomes

Purpose: The identification of genes and regulatory pathways by OxyS sRNA in Escherichia coli. Methods: mRNA profiles of OxyS overexpression in wild-type and knockout of hfq strains using Illumina sequencing. Ribosomal RNA depletion from total RNA was performed using Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria, Illumina) according to manufacturer protocols. Libraries for Illumina sequencing were made with the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) by the manufacturer’s protocol. RNA sequencing was performed on the Illumina HiSeq 2500 platform using a pair-end 50 bp sequencing. The sequence data for the reference genome (E. coli K-12 MG1655) was retrieved from the NCBI database. Quality-filtered reads were aligned to the reference-genome sequence using Bowtie2 software. The relative transcript abundance was measured in fragments in reads per kilobase of exon sequence per million mapped sequence reads (FPKM). All data processing were further performed by CLRNASeq V.1.00 (Chunlab, South Korea). Results: Using an optimized data analysis workflow, about 1 million sequence reads per sample to the E. coli K-12 genome were mapped and identified 4,224 and 4,303 transcripts in wild-type and hfq- strains, respectively. After TMM (Trimmed Mean of M-value) normalization and elmination of <100 reads in control and OxyS overexpression, 3,524 or 2,748 genes in hfq+ or hfq- strain, respetively, were utilized as a set to screening of candidates related to OxyS phenotype. Conclusions: Our study represents the first detailed analysis of OxyS transcriptomes in hfq+ and hfq- strains, generated by RNA-seq technology. The optimized data analysis workflows reported here could comparative investigate the expression profiles of sRNA in different background strains. We conclude that RNA-seq based transcriptome characterization of sRNAs would unravel genetic network in complex biologic functions. mRNA profiles of OxyS overexpression in wild type (WT) and hfq- were generated by deep sequencing using Illumina HiSeq 2500.