Characterisation of COMPASS activity in ageing yeast (no spike-in ChIP-seq experiments)
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ABSTRACT:
PROVIDER: PRJNA492238 | ENA |
REPOSITORIES: ENA
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Project description:Characterisation of COMPASS activity in ageing yeast
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Project description:ENCODE Spike-in ChIP-chip Experiments Keywords: ChIP-chip comparison
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Project description:Here, we showed that acute lost H3K4me3 induces rapid reduction in global transcription with progressively increase in magnitude over the time-course cooccurrence with lost all H3K4 methylations. To further determine the effects of COMPASS subunits degradation on H3K4 methylations occupancies genome-wide, we performed time-course spike-in chromatin immunoprecipitation and sequencing (ChIP-seq, also known as ChIP-Rx) analysis in both degron systems.
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Project description:Due to the lack of controlled microarray experimental data, evaluation of gene identification methods has been performed using simulated data. As the ability of these approaches to mimic actual experimental data is questionable, conclusions drawn can be misleading. We applied spike-in approaches to develop experimental benchmark data and used it to evaluate various gene identification methods. mRNA that correspond to specific spots on a microarray were obtained by in-vitro transcription of selected clones. They were then spiked in at 11 varying concentrations to a common pool of mRNA, creating a set of known differentially expressed genes. mRNA was obtained from mouse ATCC CRL1606 hybridoma cells. From self hybridization results 200 genes that consistently exhibited a log ratio value close to 0 were randomly selected across a range of intensity. In-vitro transcripts of these 200 genes were obtained from their respective clones. From RNA gel electrophoresis and test array results, 169 of the 200 transcripts were deemed successful. Experiments were then conducted by spiking these 169 transcripts to the common pool of mRNA extracted from the hybridoma cells. With exception of the spiked in genes, the experimental set-up is identical to a self hybridization. This experiment is thus different from other microarray experiments as the set of differentially expressed genes is known as they have been artificially created. Spiked in genes were introduced at 11 concentrations: 0pmol (self hybridization), 0.025pmol, 0.05pmol, 0.10pmol, 0.15pmol, 0.20pmol, 0.25pmol, 0.5pmol, 0.75pmol 1pmol and 2pmol. For concentrations 0pmol, 0.05pmol, 0.10pmol, 0.25pmol, 0.50pmol and 0.75pmol, 6 replicates were obtained. For concentrations 0.025pmol, 0.15pmol, 0.20pmol, 1pmol and 2pmol, 3 replicates were obtained. Dye swaps were not performed for these experiments. Keywords: other
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Project description:Due to the lack of controlled microarray experimental data, evaluation of gene identification methods has been performed using simulated data. As the ability of these approaches to mimic actual experimental data is questionable, conclusions drawn can be misleading. We applied spike-in approaches to develop experimental benchmark data and used it to evaluate various gene identification methods. mRNA that correspond to specific spots on a microarray were obtained by in-vitro transcription of selected clones. They were then spiked in at 11 varying concentrations to a common pool of mRNA, creating a set of known differentially expressed genes. mRNA was obtained from mouse ATCC CRL1606 hybridoma cells. From self hybridization results 200 genes that consistently exhibited a log ratio value close to 0 were randomly selected across a range of intensity. In-vitro transcripts of these 200 genes were obtained from their respective clones. From RNA gel electrophoresis and test array results, 169 of the 200 transcripts were deemed successful. Experiments were then conducted by spiking these 169 transcripts to the common pool of mRNA extracted from the hybridoma cells. With exception of the spiked in genes, the experimental set-up is identical to a self hybridization. This experiment is thus different from other microarray experiments as the set of differentially expressed genes is known as they have been artificially created. Spiked in genes were introduced at 11 concentrations: 0pmol (self hybridization), 0.025pmol, 0.05pmol, 0.10pmol, 0.15pmol, 0.20pmol, 0.25pmol, 0.5pmol, 0.75pmol 1pmol and 2pmol. For concentrations 0pmol, 0.05pmol, 0.10pmol, 0.25pmol, 0.50pmol and 0.75pmol, 6 replicates were obtained. For concentrations 0.025pmol, 0.15pmol, 0.20pmol, 1pmol and 2pmol, 3 replicates were obtained. Dye swaps were not performed for these experiments.
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