Computational protocol: Small Toxic Protein Encoded on Chromosome VII of Saccharomyces cerevisiae

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Protocol publication

[…] Yeast cells that harbored pTOWug2–836, pTOW40836, and pTOW-Rear2 were cultivated in SC—Ura medium until the mid-log phase, and RNA from each culture was then isolated using the hot phenol method []. A cDNA library was prepared using a SureSelect strand-specific RNA library preparation kit (G9691A, Agilent), and sequencing was performed using an Illumina Hiseq2500 with TruSeq SBS kit v3-HS. The software connected to GenomeSpace (http://www.genomespace.org) was used for the sequence data analysis. The sequence data were analyzed using TopHat (ver. 6) and Cufflink/cuffdiff (ver.4) on the GenePattern platform (http://genepattern.broadinstitute.org), with sacCer3 for gene annotation (http://genome.ucsc.edu/cgi-bin/hgTables). First, we isolated genes that differed significantly (FDR < 0.5) between pTOWug2–836 and pTOW-Rear2 (Comp1), pTOW40836 and pTOW-Rear2 (Comp2), pTOWug2–836 and pTOW40836 (Comp3), and the pTOW-Rear2 duplicates (Comp4). Next, we prepared a gene list from genes isolated in Comp1 or Comp2, but not in Comp3 or Comp4 (summarized in . and ). The Integrative Genomics Viewer (IGV2.3, http://www.broadinstitute.org/igv/) was used to visualize the sequence reads shown in . The GO, publication, and pathway enrichments were analyzed using YeastMine (http://yeastmine.yeastgenome.org). […]

Pipeline specifications

Software tools TopHat, Cufflinks, GenePattern, IGV
Application RNA-seq analysis
Organisms Saccharomyces cerevisiae, Saccharomyces paradoxus
Chemicals Amino Acids, Ergosterol