Project description:We profiled the time-dependent expression levels of a set of 95 genomically characterized haploid yeast segregants in response to rapamycin. They were derived from two genetically diverse parental yeast strains, BY4716 and RM11. Both parental strains have been sequenced. Our goal is to capture widespread transcriptional changes over time, and to leverage on both the temporal and genetic variations to construct regulatory networks.
Project description:Genotyping pools of wildtype(wt) or sup35-C653R(sup35) BYxRM segregants after growth on YPD (control) or YPD+drug-of-interest(selection) 10 Growth conditions tested are control (YPD alone), and YPD plus the following drugs: Chlorpromazine, CoCl2, Cycloheximide, Diamide, E6berbamine, Ethanol, H2O2, Neomucin, and Tunicamycin. Two-condition experiment, control vs selection. 6 biological replicates for control and two biological replicates for each selection included.
Project description:To test the role of Mpe1 in transcription termination we inserted a mini-auxin induced degron (mAID) at the C-terminal end of the endogenous MPE1 locus in the yeast Saccharomyces cerevisiae. We treated Mpe1-mAID cells (JRY101) or wild type cells (WT, YMK728) with 1 mM auxin for 30 minutes and labeled the nascent RNA with 4-thiouracil (4tU) for 6 minutes. The nascent RNA fraction was prepared by biotinylating the 4tU-labeled RNA followed purification with streptavidin beads. Nascent and total RNA fractions were depleted of ribosomal RNA and strand-specific libraries prepared. Libraries were single-end sequenced using an Ilummina HiSeq 4000 instrument.
Project description:Data from Excision-seq experiments to map deoxyuridine and pyrimidine dimers in S. cerevisiae and E. coli For uracil mapping in pre-digestion Excision-seq, uracil was excised from genomic DNA from dut ung yeast and bacteria yielding double-stranded DNA fragments. Adapters were ligated to these fragments for Illumina library preparation. Using this method, the number of reads at a genomic location corresponds to the quantity of dU at that location. In post-digestion Excision-seq, uracil-containing library fragments are destroyed by UDG treatment, thus coverage is inversely proportional to uracil content. For pyrimidine dimer Excision-seq, yeast were irradiated with high doses of UVC light to generate a large number of DNA modifications. Using the excision repair enzyme UVDE from S. pombe, we excised pyrimidine dimers, cutting the phosphodieseter bond to release small double stranded fragments. These fragments were repaired with either CPD photolyase or 6-4 photolyase to generate independent libraries for each modification type. Illumina adapters were ligated to these fragments for library preparation. Using this method the number of reads at a genomic location corresponds to the quantity of the 3' pyrimidine of the dimer.
Project description:We investigate if the differences in phenotype and transcriptome over age might be explained by an underlying change on the epigenetic level. We performed single-cell ATAC sequencing using the 10x Chromium platform. We profiled 2259 nuclei prepared from 3 young liver tissues and 2490 nuclei from 3 old liver tissues.
Project description:We investigate if the differences in phenotype and transcriptome over age might be explained by an underlying change on the epigenetic level. We performed single-cell ATAC sequencing using the 10x Chromium platform. We profiled 4838 nuclei prepared from 3 young liver tissues and 3361 nuclei from 3 old liver tissues.
Project description:Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results. Genomic input (labelled with Cy3) and IP'ed DNA (labelled with Cy5) using a MYC Ab of either DNA Pol2-13MYC or Rrm3-MYC from asynchronously grown S. cerevisiae cells in rich media were hybridized to whole-genome PCR-based arrays containing ORF and intergenic regions of the entire genome (Ivery et al 2001). At least three biological replication and one technical replicate (dye swap) were performed. Log2 transformed median normalized ratios (IP/IN) were averaged for each experiment and significant peaks of either DNA Pol2 or Rrm3 association were identified
Project description:Cell division is a highly regulated process that secures the generation of healthy progeny in all organisms, from yeast to human. Dysregulation of this process can lead to uncontrolled cell proliferation and genomic instability, both which are hallmarks of cancer. Cell cycle progression is dictated by a complex network of kinases and phosphatases. These enzymes act on their substrates in a highly specific temporal manner ensuring that the process of cell division is unidirectional and irreversible. Key events of the cell cycle, such as duplication of genetic material and its redistribution to daughter cells, occur in S-phase and mitosis, respectively. Deciphering the dynamics of phosphorylation/dephosphorylation events during these cell cycle phases is important. Here we showcase a quantitative proteomic and phosphoproteomic mass spectrometry dataset that profiles both early and late phosphorylation events and associated proteome alterations that occur during S-phase and mitotic arrest in the model organism S. cerevisiae. This dataset is of broad interest as the molecular mechanisms governing cell cycle progression are conserved throughout evolution.
Project description:Capture and massively parallel DNA sequencing of ribonucleotides embedded in S. cerevisiae genomic DNA We developed a new method to map the positions of ribonucleotides embedded in DNA using the unique specificity of A. thaliana tRNA ligase. Ribonucleotides were generated in budding yeasts of different genetic backgrounds and mapped to single nucleotide resolution using the new method.
Project description:Breast cancer is the most prevalent cancer in women 1, and most cases are believed to have a sporadic, rather than heritable basis 2. To identify breast cancer driver genes, we exploited the C3H-Mcm4Chaos3/Chaos3 (“Chaos3”) mouse model that, by virtue of bearing a defective DNA replicative helicase subunit that causes elevated genomic instability (GIN), sustains somatic alterations ultimately causing mammary adenocarcinomas 6. Array Comparative Genomic Hybridization (aCGH) analysis of Chaos3 mammary tumors revealed recurrent copy number alterations (CNAs), most notably deletion of the Neurofibromin 1 (Nf1) tumor suppressor gene in all cases. NF1, a negative regulator of RAS, is traditionally recognized for its role in driving the development of neurofibromas in the context of the human disease Neurofibromatosis Type 1, but not breast cancer. Genomic DNA from tumor and reference samples were hybridized to NimbleGen 3x720K mouse CGH arrays. Two reference samples were used independently. CNAs were visualized using Nimblegen, IGV, and KCsmart software 32. Select genes were validated via qPCR. Critical regions within each Chaos3 CNA were identified as the region with the greatest overlap across multiple Chaos3 tumors. Recurring Copy Number Variations (CNVs) for 12 Chaos3 tumors and 2 MMTV-Neu mammary tumors analyzed by aCGH are indicated. Samples analyzed are primary tumors except where indicated.