Project description:To identify the genome-wide transcriptional changes that occur throughout sexual developmen of C. neoformans, we conducted a time-course microarray experiment spanning six developmental stages to generate a temporal expression pattern for each known gene. C. neoformans a x α co-cultures were grown under sexual development conditions (V8 agar), and RNA was harvested at 0.5, 6, 12, 24, 48, and 72 hours post-mixing. Time points were chosen based on microscopic identification of cell types of interest: fusants, filaments (early and late), basidia, and spores (Figure 1B). Microarray hybridizations were conducted in a loop design, with each sample serving as a reference for the following time point in the experiment (e.g. 0.5 hours vs. 6 hours, 6 hours vs. 12 hours, etc).
Project description:To identify the genome-wide transcriptional changes that occur throughout sexual developmen of C. neoformans, we conducted a time-course microarray experiment spanning six developmental stages to generate a temporal expression pattern for each known gene. C. neoformans a x M-NM-1 co-cultures were grown under sexual development conditions (V8 agar), and RNA was harvested at 0.5, 6, 12, 24, 48, and 72 hours post-mixing. Time points were chosen based on microscopic identification of cell types of interest: fusants, filaments (early and late), basidia, and spores (Figure 1B). Microarray hybridizations were conducted in a loop design, with each sample serving as a reference for the following time point in the experiment (e.g. 0.5 hours vs. 6 hours, 6 hours vs. 12 hours, etc). The experiment spanned six time points of development: 0.5 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours. Hybridizations were conducted in a loop design, with each sample serving as the reference for the consecutive time point - resulting in 5 experimental comparisons: .5hr vs 6 hr, 6 hrs vs 12 hrs, 12 hrs vs 24 hrs, 24 hrs vs 48 hrs, and 48 hrs vs 72 hrs. Each comparison consistent of duplicate hybridizations with dye swaps. Arrays contain duplicate genomes, resulting in eight-fold coverage within each comparison. (total of 20 hybridizations)
Project description:To identify the genome-wide transcriptional changes that occur throughout germination of C. neoformans spores, we conducted a time-course microarray experiment spanning six timepoints to generate a temporal expression pattern for each known gene. Spores were placed in rich medium and allowed to germinate for 10 hours, when they start to replicate as yeast. Each time point was flash frozen in liquid nitrogen and RNA was harvested for each time point at the same time. Microarray hybridizations were conducted in a reference pool design, where each time point was mixed together in equal amounts to make a reference pool sample. Then each time point was hybrdized against the reference pool. Characterization of the genes and pathways that are regulated during germination of this ubiquitous fungal pathogen will allow us to better understand how infectious spores resume vegetative growth, a process that likely is critical for interaction between C. neoformans and a host.