Computational protocol: Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans

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Protocol publication

[…] For RNA-Seq, YNBNC medium was inoculated with 5 × 107 cells per ml as described above, and cultures were grown for 4 h at 37°C. Cells were pelleted by centrifugation at 4,500 × g for 5 min and snap-frozen in liquid nitrogen. RNA was extracted with the Epicentre MasterPure yeast RNA purification kit (MPY03010). RNA quality and quantity were assessed by using a fragment analyzer (Advanced Analytical, Ankeny, IA) and Qubit (Invitrogen, Carlsbad, CA), respectively. mRNA was enriched by hybridization to oligo(dT) beads. Directional RNA-Seq libraries were prepared with TruSeq stranded mRNA library prep chemistry with unique TruSeq indices, using an automated liquid-handling system. Libraries were pooled and sequenced on a NextSeq500 instrument, using 2× 75-bp paired-end sequencing (a high-output flow cell). Raw reads were processed using the CLC Genomics Workbench platform (v. 8.5.1) and the default parameter settings installed by the manufacturer. All sequences were trimmed and mapped to the SC5314 reference genome (version A21-s02-m09-r04; http://www.candidagenome.org) and with the use of the RNA-Seq analysis tool, and mapped reads were normalized to control for any differences in library size by using the commands “calcNormFactors,” “estimateCommonDisp,” and “estimateTagwiseDisp” with default settings in the edgeR package (v. 3.14.0). The full Gene Ontology annotation was used for GO term analysis. The gene association file, created 19 September 2016, was downloaded from the CGD website (http://www.candidagenome.org), and only annotations assigned to C. albicans (taxon 5476) were used. In total, 6,313 unique genes had at least one associated GO term and served as the background distribution of observed gene ontologies for the C. albicans genome in this study. GO enrichment analysis of the comparisons between the WT and the cyr1 mutant at pH 4 and pH 7 was evaluated using an R script (GOstats.R, within bioconductor), in which the GSEAGOHyperGParams function was used for calculating a Bonferroni-corrected P value with a cutoff of 0.05 to determine significant GO term enrichment in the categories Biological Process, Cellular Component, and Molecular Function. The 100 most significantly enriched terms were retained for analysis, followed by removal of similar terms. […]

Pipeline specifications

Software tools CLC Genomics Workbench, edgeR, GOstats
Application RNA-seq analysis
Organisms Candida albicans, Saccharomyces cerevisiae, Homo sapiens
Diseases Candidiasis, Infection
Chemicals Cyclic AMP, Guanosine Triphosphate, Fluconazole