Computational protocol: Integration of the tricarboxylic acid (TCA) cycle with cAMP signaling and Sfl2 pathways in the regulation of CO2 sensing and hyphal development in Candida albicans

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Protocol publication

[…] Cells of the WT and sfl2/sfl2 mutant were first grown at 25°C in liquid Lee’s glucose medium for 24 hours, then spread onto YPD medium plates and incubated at 37°C in air or in 5% CO2 for 22 hours. Colonies were harvested and total RNA was extracted as described above. RNA-Seq analysis was performed by the company Berry Genomics Co. (Beijing). Approximately 10 million (M) reads were sequenced in each library of the samples. Briefly, mRNA was purified from total RNA using Oligo(dT) magnetic beads, and fragmented into small pieces (200–700 bp). The cleaved RNA fragments were primed with random hexamers and used to synthesize the first-strand and second-strand cDNA. Sequencing adapters were ligated to the cDNA fragments. The library products were then sequenced using an Illumina HiSeq 2500 V4. Illumina software OLB_1.9.4 was used for base-calling. The raw reads were filtered by removing the adapter and low quality reads (the percentage of low quality bases with a quality value ≤3 was >50% in a read). Clean reads were mapped to the genome of C. albicans SC5314 using TopHat (version2.1.1) and Cufflinks (version2.2.1) software []. Mismatches less than two bases were allowed in the alignments. Relative gene expression levelswere calculated using the FPKM (Fragments Per kb per Million reads)method; FPKM = 106C/(NL/103), where “C” is the number of fragments that uniquely aligned to gene A, “N” represents the total number of fragments that uniquely aligned to all genes, and “L” is the number of bases of gene A. GO functional enrichment analysis was carried out according to GO terminology determined using the online CGD GO Term Finder tool (http://www.candidagenome.org/cgi-bin/GO/goTermFinder). […]

Pipeline specifications

Software tools TopHat, Cufflinks, GO::TermFinder
Application RNA-seq analysis
Organisms Homo sapiens, Candida albicans
Chemicals Adenosine Triphosphate, Carbon, Carbon Dioxide