How host-like signals drive gene expression and gene expression drives capsule growth in Cryptococcus neoformans [ep_pert]

Cryptococcus neoformans is a fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew wild-type cells and cells lacking components of the cAMP pathway: Pde1, Pde2, Pkr1. We also took samples for RNA-Seq at 30, 90, 180, 1440 minutes and carried out RNA-Seq in quadruplicate. We also measured capsule thickness at 1440 min. After growth in YPD at 30C, WT cells and cells lacking PDE1, PDE2, or PKR1 were washed in PBS and transferred to RPMI medium at 30C. Samples were taken for RNA-Seq at 0, 30, 90, 180, 1440 minutes after transfer. We aimed for at least four replicates of each concentration at each time point. Replicates were grown and library-prepped on separate days to account for day-to-day variability. On each growth and library prep date, we included cells from between the PBS wash and innocoulation into the planned growth conditions, as a constant control and an initial timepoint. These PBS samples are labeled as time 0. However, they turned out to have much more variability in their RNA-Seq data than other conditions, so we did not use them in our analysis.