RNA-Seq of Candida albicans: grown in rich media
Yeast cultures were grown in either rich or minimal media and sampled at multiple time points corresponding to 2, 5, 8, 11, 24, and 48 hours post-inoculation. At each time point, cell pellets were collected by centrifugation and immediately flash-frozen in liquid nitrogen for RNA extraction. Total RNA was extracted using a standard acid-phenol chloroform method, followed by DNase treatment to remove genomic DNA contamination. RNA integrity was assessed using a Bioanalyzer or TapeStation. Libraries were prepared from high-quality RNA using a strand-specific protocol and sequenced on an Illumina platform to generate paired-end FASTQ files. Raw FASTQ reads were subjected to quality control, including adapter trimming and filtering of low-quality reads using tools such as FastQC and Trimmomatic. Cleaned reads were aligned to the reference genome using a splice-aware aligner (STAR), and read counts per gene were quantified using featureCounts to generate a raw count matrix. Gene expression data were analyzed using the DESeq2 package in R. A generalized linear model was fitted using the formula ~ Time + Media to account for both temporal and media-dependent effects on gene expression. Normalization and statistical testing were performed according to DESeq2 guidelines. Hierarchical clustering was used to group genes by temporal expression profiles. Genes with similar expression dynamics were subjected to Gene Ontology (GO) term enrichment analysis using OrgDB annotations sourced from UniProt and Candida Genome Database association files. Enrichment was evaluated for overrepresented biological processes using over-representation analysis.