Direct RNA Sequencing and Transcriptomic Analysis of Pseudomonas putidaPublic
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The transcriptome of a cell is the total messenger RNA (mRNA) content of the cell. The transcriptome is useful to understand the changes that can occur in gene expression, specifically amongst growth conditions. Gene expression can be analyzed through many sequencing techniques. One technique is direct RNA sequencing (direct RNA-seq), a relatively new technique that does not need RNA to be converted to cDNA and does not rely on amplification methods of any kind. Direct RNA-seq has not been utilized to perform analysis on the transcriptome in the soil environment. Typically, this method is not the preferred method for bacterial RNA sequencing, mostly due to the need for rRNA depletion, lowering the concentration of pure RNA samples by at least 90%. Direct RNA-seq was attempted to investigate the transcriptome of Pseudomonas putida in various growth conditions. This project was completed in hopes that the transcriptomic data acquired would be used to engineer improved biosensors for environmental applications. After several attempts at isolating viable RNA for sequencing, it was determined that the concentration of the samples acquired was too low for rRNA depletion, making it impossible to sequence. Instead, we analyzed previously acquired transcriptomic data of species native to the soil environment to find any similarities amongst them. Our paper presents the attempted methods used to isolate RNA, along with our recommendations for further projects to consider.
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