The objective of this experiment was to study the mechanism of de novo gene birth in Saccharomyces cerevisiae. While the model organism S. cerevisiae has one of the highest quality reference genomes and annotations, other members of Saccharomycotina are not so fortunate. We designed our experiment to compare the transcriptomes of 11 species of yeast in ‘normal’ as well as ‘stress’ conditions as it has been hypothesized that de novo genes could play a role in the evolution of environmental stress responses. We used this RNA sequencing data to assemble reference-independent transcriptomes for each species to avoid biases in our comparative transcriptomic analysis caused by varying qualities of reference annotations.</p><p>We grew 11 species (S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, N. castelii, K. lactis, L. waltii, L. thermotolerans, L. kluyveri, and Schizo. pombe) in two parallel conditions:</p><p>1. Rich medium- We isolated isogenic populations from streaked plates, grew cultures overnight in culture tubes, then inoculated 20mL of a custom rich medium in 50mL Erlenmeyer flasks at 30ºC. Cells were harvested in log growth phase at an OD600 of approximately 0.25, then subsequently frozen at -80ºC.</p><p>2. Oxidative stress- The same isogenic populations were grown in parallel, identical to the first ‘Rich medium’ condition however, 30 minutes prior to harvesting the cells, diluted hydrogen peroxide was added to a final concentration of 1.5mM.</p><p> The custom rich medium, taken from the following study, was designed to accommodate growth of many species of yeast: Tsankov AM, Thompson DA, Socha A, Regev A, Rando OJ. “The role of nucleosome positioning in the evolution of gene regulation”. PLoS Biol 2010. doi.org/10.1371/journal.pbio.1000414