Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Saccharomyces paradoxus genes Overall design: Total RNA was collected in mid-log phase from Saccharomyces paradoxus cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Saccharomyces paradoxus.
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Saccharomyces paradoxus genes Total RNA was collected in mid-log phase from Saccharomyces paradoxus cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Saccharomyces paradoxus.
Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions. Transcriptomes of two yeast species under mid-log phase, early stationary phase, and after heat shock treatment were generated by Illumina HiSeq 2000 paired-end sequencing
Project description:The ability to perform complex bioassays in parallel enables experiments otherwise impossible due to throughput and cost constraints. By way of example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is barcoded with unique DNA sequences. It is, however, time consuming and expensive to individually barcode individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique barcodes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of barcoded DAmP (Decreased Abundance by mRNA Perturbation) loss-of-function strains comprising 87.1% of all essential yeast genes. This test collection validates both the Barcoders and the DAmP collection as useful tools for genome-wide chemical genetic assays.
Project description:We determined nucleosome positions genome-wide in diploid Saccharomyces species undergoing early stages of synchronous meiosis. This study sought to assess if meiotic DNA double-strand break formation occurred preferentially in promoter nucleosome-depleted regions in other Saccharomyces species, as it does in S. cerevisiae SK1 (Pan et al. 2011 Cell 144:719-731). Overall design: Twelve samples were sequenced: three samples of S. cerevisiae YPS128 (two biological replicates, and a technical replicate digested with different amount of MNase), two biological replicates of S. cerevisiae UWOPS03-461.4, three samples of S. paradoxus (two biological replicates, and a technical replicate digested with different amount of Mnase), two biological replicates of S. mikatae, two biological replicates of S. kudriavzevii.