Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1280644 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:During HIV-1 assembly, the structural protein Pr55Gag specifically selects viral genomic RNA by interacting with packaging signals present in the 5’ untranslated region (UTR). Despite a large number of studies, Pr55Gag binding to the genomic RNA remains poorly understood. To precisely define the Pr55Gag binding site we performed Mutational Interference Mapping Experiment on the 5’UTR and the beginning of the gag gene. To do this, we first generated mutant RNA libraries by in vitro transcription of PCR products that had been subjected to two or three sequential rounds of mutagenic PCR. Sequencing showed a mutation rate of 0.6% ensuring that the vast majority of molecules contained at least one mutation. RNA was mixed with His-tagged Pr55Gag, and functional selection was performed using magnetic beads to separate the bound and the unbound RNA populations. RNA was then reverse transcribed, converted into double stranded DNA, and sequenced using the Illumina HiSeq 2000 platform. This platform generates a large number of sequencing reads but with a read length that is shorter than our target sequence (100 nucleotides from each end of the DNA versus 532 nucleotides). We overcame this limitation by randomly fragmenting our DNA into fragments ranging from 100 to 500 nucleotides during library preparation. Using this strategy sequencing data was obtained for all positions of the target, whilst also retaining linkage data for all pairs of nucleotides whose distance does not exceed the largest fragment size. We aligned approximately 170 million sequences to the reference genome, comprising ~9x107 mutations over ~3x1010 base pairs, after quality control steps. We also sequenced cDNA from a non-mutated template, which we used in a mathematical analysis to correct for errors introduced during in vitro transcription, library preparation and sequencing. Mutational Interference Mapping Experiment of Pr55Gag binding to RNA in vitro
2015-06-13 | E-GEOD-69805 | biostudies-arrayexpress
Project description:This data set represents the results of two reverse labeled experiments from wild-type and RRP6delta S. cerevisiae that has been hybed to arrays containing PCR products for ORFs and Intergenic Features Keywords: genetic modification
2006-01-15 | GSE3813 | GEO
Project description:In vitro batch fermentation by-products
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Project description:Combination of reverse- and chemical genetic screens reveals a network of novel angiogenesis inhibitors and targets Drug target identification and validation are bottlenecks in the drug discovery process. Accordingly there is a need to develop new methods to facilitate the development of much-needed innovative drugs. We have combined reverse- and chemical genetics to identify new targets modulating blood vessel development. Through mRNA expression profiling in mice we identified 155 drugable gene products that were enriched in the microvasculature. Orthologs of 50 of these candidates were knocked down in a reverse genetic screen in zebrafish. 16 of the 50 genes encoded products that affected angiogenesis. In parallel, screening of 300 known drugs and pharmacologically active compounds in a human cell-based angiogenesis assay identified 11 angiogenesis inhibitors. Strikingly the reverse- and chemical genetic screens identified an overlap of three gene products of the same superfamily of serine/threonine protein phosphatases and two compounds targeting that family. Furthermore, the gene products identified in the reverse genetic screen comprise an interacting network with the targets of the chemical genetic screen. Thus, combining reverse- and chemical genetic screens is a powerful approach to identify novel biological processes and drug targets in vertebrates. Keywords: Cell-type comparison
2009-12-10 | GSE10035 | GEO
Project description:Nucleosomal DNA was prepared using Simple ChIP Enzymatic Chromatin IP Kit according to manufacturer’s instruction. Briefly, Nuclei were isolated from purified Ter119 negative or in vitro cultured erythroblasts. Cross-linked native chromatin was then digested with MNase into mononucleosomal DNA. Sequencing libraries were generated from nucleosomal DNA, and sequencing was carried out using the Illumina system according to the manufacturer’s specification. In this study, we purified chromatin from in vitro cultured mouse fetal liver erythroblasts on day 0, day 1, and day 2. The chromatins were digested by micrococcal nuclease to make mononucleosomal products, which were further analyzed by next generation sequencing analysis. We aim to determine the dynamic changes of nucleosome during terminal erythropoiesis.
2014-10-01 | E-GEOD-55825 | biostudies-arrayexpress
Project description:Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II transcription sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions rather than to autonomous RNA genes. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes. Chromatin immunoprecipitation of RNA polymerase II phosphorylated in ser5 followed by multiparallel sequencing was performed in bone marrow-derived macrophages. The experiment was also carried out in cells treated for 2hrs with lipopolysaccharide (LPS).
2010-05-21 | E-GEOD-19991 | biostudies-arrayexpress