Candida albicans smFISH data processing pipeline

Data processing pipeline connected to the publication: "Single-molecule Fluorescent In Situ Hybridization (smFISH) for RNA detection in the fungal pathogen Candida albicans"

Abstract Candida albicans is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of C. albicans to reversibly change morphology and to grow as yeast, pseudohyphal or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help fight C. albicans infections. While numerous studies investigated gene expression changes occurring during C. albicans morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single molecule fluorescent in situ hybridization (smFISH) protocol for C. albicans, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae or hyphal cells. We quantified the expression of two mRNAs, cell cycle-controlled mRNA (CLB2) and a transcription regulator (EFG1), which show differential expression in the different morphological cell types and in different nutrients conditions. In this protocol we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell-segmentation and mRNA spot analysis. Raw data is provided with the protocol to favor reproducibility. This approach could benefit the molecular characterization of C. albicans and other filamentous fungi, pathogenic or non-pathogenic.