Design a hypothetical experiment and write a short prompt to describe how you would go about conducting such an experiment from start to finish, including how you would grow cells, how to prepare them for RNA-seq, and what you would do after you have obtained raw gene counts. Please include step by step of your experiment. Example: I work in a microbial bioengineering lab which focuses on production of metabolites with high-added value. Our lab currently has established media on which our microbe grows; however, we have 5 experimental growth substrates which we predict can give us better yields, in comparison to our standard media. The way I would approach this scenario is growing our bacterial strain, in triplicate, on our standard media and all 5 experimental growth substrates. I would ensure all other factors remain static (growth time, growth temperature, amount of biomass used to inoculate) and harvest cells at the end of their incubation. After harvesting cells, I would extract mRNA using an established kit and prepare samples for cDNA library construction/RNA sequencing. After I receive my raw FASTQ reads, I would either use an established bioinformatic pipeline or an existing pipeline to obtain raw gene counts, at which point I would conduct differential gene expression analysis with DESeq2 to get an overview of which genes are up- or down-regulated, relative to our standard growth media. Subsequently, I suspect that genes in the TCA cycle and pentose phosphate pathway (PPP) will be the most impacted; I take the results of my differential gene expression analysis and use them as input for pathView to check which enzymes/enzymatic reactions are being influenced, relative to our standard growth media.