Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.
Project description:Targeted paired-end sequencing of cDNA from unfragmented nascent RNA from exponentially growing S. cerevisiae cells was employed to obtain Pol II transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010) or enriched from total RNA with polyadenylated RNA depletion. The nascent 3â end was labeled with a 3â DNA adaptor through ligation. A PCR with a forward primer in the first exon of select intron-containing genes amplifies nascent transcripts of specific genes and ensures sequencing adaptor attachment for paired-end sequencing. With this approach co-transcriptional splicing progression with distance from the intron end could be analyzed for 87 genes. Note that the unmapped and mapped data also include genes that did not pass the read coverage requirements in SMIT analysis. Nascent RNA profiles for mainly intron-containing genes were generated with paired-end sequencing with Illumina HiSeq technology.
Project description:This experiment was performed in order to assess the specificity of Rad9 binding to S. cerevisiae genome. In another ChIP-chip experiment in SC BCS BPS growth conditions we have found Rad9 present to a significant number of genomic loci with a bias to transcriptionally active regions. In order to see if that pattern was random and depended or not on the activity state of the genes, we conducted a Rad9 ChIP on chip experiment with the strain grown in medium with galactose instead of glucose, where it is known that particular gene groups are transcriptionally activated. We then compared to the results of our previous experiment where the strain was grown in glucose.
Project description:Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states. Keywords: Chip-chip, time course, histone turnover Ratios between Gal-induced H3-Flag and constitutive H3-Myc at 8 time points for unsynchronized yeast. Hybridization to high-resolution printed arrays of ~4% of the yeast genome.
Project description:Cohesin acetylation by Eco1 during DNA replication establishes sister chromatid cohesion. We show that acetylation makes cohesin resistant to Wapl activity from S-phase until mitosis. Wapl turns out to be a key regulator of cohesin dynamics on chromosomes by controling cohesin maintenance following its establishment in S-phase and its role in chromosome condensation. The Affymetrix Saccharomyces cerevisiae Chip Tiling 1.0R Arrays were used to analyze the binding pattern of Scc1 along the genome of Saccharomyces cerevisiae in late G1 and metaphase arrested cells.
Project description:Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states. Keywords: Chip-chip, time course, histone turnover Ratios between Gal-induced H3-Flag and constitutive H3-Myc at 5 time points for G1-arrested yeast. Hybridization to high-resolution printed arrays of ~4% of the yeast genome.
Project description:Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3M-bM-^@M-^Y major domain of the 20S pre-ribosomal RNA. Three datasets of yeast ribosomal samples subjected to different chemical modifications; 1M7 dataset contains 8 different modified samples and 2 control samples; NAI dataset contains 3 different modified samples and 2 control samples; DMS dataset contains 1 modified sample and 1 control sample. Each sample consists of at least two replicates.