Genomewide measurements of whole embryo mRNA decay in Drosophila
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ABSTRACT: whole embryo (all tissues) measurement of mRNA decay by 4-thiouridine pulse-chase These TU-Decay microarrays analyze mRNA levels at three timepoints: a one hour pulse, one hour chase, and three hour chase. Measurements with or without transcription inhibition by actinomycin D (ActD) were compared.
Project description:Neural-specific mRNA decay measurements by TU-Decay technique in control and Pumilio knockdown embryos These TU-Decay microarrays analyze mRNA levels at three timepoints: a one hour pulse, one hour chase, and three hour chase. Neural-specific RNA purification was achieved using prospero-GAL4 driving UAS-T.g.UPRT. Pumilio knockdown in the nervous system was acheived using UAS-Pum(RNAi) driven by prospero-Gal4.
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. Keywords: RNA_stability_design
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. User Defined
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase.
Project description:To study RNA degradation by RNA-seq, global inhibition of transcription has to be performed in order to measure RNA decay with no interference from RNA sysnthesis. However, transcription inhibitors are generally toxic, can affect abundance many transcripts, and cause delay in RNA decay by residual RNA synthesis. We envisioned that AIR-seq, in conjunction with the pulse-chase labeling, would enable genome-wide analysis of RNA degradation in E. coli with no need of transcription inhibition.
Project description:Human articular chondrocytes were isolated from normal or osteoarthritic tissue. RNA decay was measured across the transcriptome in these cells by microarray analysis following an actinomycin D chase for 0, 1, 3 and 5 hours. Normalisation was conducted by quantile normalising each set of four decay curve points (i.e. 0, 1, 3 and 5 hour samples for a given donor's cells) independently of the other data. This meant that each decay curve is normalised independently of the others.
Project description:A Brunello CRISPR-Cas9 library screen was performed with OVCAR3 and CCL218 cells in the presence or absence of hydroxychloroquine. Drug pulse chases were used, with 48h of drug exposure followed by replacement of fresh media. Cells were allowed to grow to near confluence and then another drug pulse-chase was performed. After each pulse-chase, a pool of cells were collected for sgRNA quantitation. Three pulse chases were performed. Cell pellets were processed for genomic DNA and sgRNA sequences amplified by PCR for NGS-based quantitation. Once quantified by NGS analysis, MAGeCK MLE analysis was performed to determine which sgRNAs were most associated with resistance or sensitization to hydroxychloroquine.