Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Method: HaCaTs and primary keratinocytes were infected with WT USA300 S. aureus (MOI 100:1) for 1 hour before cell lysates were prepared. Poly-A pull-down was used to enrich mRNAs from total RNA samples (200ng-1ug per sample, RIN>8 required) and to proceed to library preparation by using Illumina TruSeq RNA prep kit. Libraries were then sequenced using Illumina HiSeq2500 at Columbia Genome Center. The samples were multiplexed in each lane, which yields targeted number of singleend/paired-end 100bp reads for each sample, as a fraction of 180 million reads for the whole lane. The raw read files were not available for submission because they have been deleted by the core facility. Results: Using an optimized data analysis workflow, we identified 15,780 transcripts in the skin of both HEKn and HaCaT cells. Expression values were analyzed using Ingenuity Pathway Analysis (IPA). Conclusions: Our study represents the first detailed analysis of HEKn and HaCaT transcriptomes, with biologic replicates, generated by RNA-seq technology after WT MRSA USA300 infection. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.
Project description:Identify the function of pE66L Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Nrl-- Retinal Transcriptomes
