Analysis of RNA-TAG labeling in HeLa lysates
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE149274 | GEO | 2020/04/25
REPOSITORIES: GEO
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Project description:Analysis of RNA-TAG labeling in HeLa lysates
| PRJNA627877 | ENA
Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2009-12-08 | GSE18898 | GEO
Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.
2010-05-19 | E-GEOD-18898 | biostudies-arrayexpress
Project description:The importance of RNA in regulating cellular processes has generated tremendous interest in methods to reliably characterize RNAs and their interactions within cells. Existing methodologies rely on noncovalent interactions to facilitate RNA affinity purification, hindering the purification of less abundant transcripts and limiting the stringency of the purification conditions. Here we demonstrate that enzymatic installation of a single covalent biotin modification directly onto an RNA of interest enables robust affinity purification of RNA-protein complexes via immunoblotting or quantitative mass spectrometry. Using this approach, known binding partners of 7SK snRNA, an important regulatory RNA, were successfully identified. This approach was further applied to the study of the long-noncoding RNA HOTAIR, using a modification of existing hairpin structures to allow for RNA-TAG labeling. A 4-nucleotide internal mutation enabled RNA-TAG mediated identification of putative HOTAIR binding partners in MCF7 breast cancer cells. Lastly, the selective and efficient labeling of an expressed RNA in mammalian cell lysate was demonstrated using a dimerized TGT enzyme through quantitative PCR and RNA-sequencing, enabling 145-fold direct enrichment from mammalian cell lysates. The flexibility and breadth of the RNA-TAG approach suggest that this methodology could be routinely applied to study RNA-protein complexes with minimal perturbation of the RNA sequence and to target less abundant RNA transcripts.
2020-03-13 | MSV000085100 | MassIVE
Project description:Hox protein transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. Understanding the basis of Hox function has long faced the problem that, while the different members of the Hox family show clear functional specificity in vivo, they all show very similar binding specificity in vitro. Based on in vitro studies, Hox cofactors may increase Hox binding selectivity but a satisfactory understanding of in vivo Hox target selectivity is still lacking. We have carried out a systematic analysis of the in vivo genomic binding profiles of all eight Drosophila Hox proteins using transient transfection in Kc167 cells to examine Hox protein targeting. We find that Hox proteins show considerable binding selectivity in vivo in the absence of the canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility; binding sites in less accessible chromatin show the highest selectivity and the different Hox proteins exhibit different propensities to bind less accessible chromatin. High Hox binding selectivity is also associated with high affinity binding regions, leading to a model where Hox proteins derive binding selectivity through an affinity-based competition with nucleosomes. Provision of the Extradenticle/Homothorax cofactors generally leads to an increase in the number of Hox binding regions and promotes the binding to regions in less accessible chromatin, however the provision of these cofactors has little effect on the overall selectivity of Hox targeting. These studies indicate that chromatin accessibility plays a key role in Hox selectivity and we propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of genomic targets for different Hox proteins. We suggest that this mechanism may also be relevant to other transcription factor families. This SuperSeries is composed of the SubSeries listed below.
2019-05-15 | GSE122575 | GEO
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of two cDNA libraries were submitted to deep sequencing: a sample from siH19-transfected cells and a control sample.
2014-12-22 | E-GEOD-62679 | biostudies-arrayexpress
Project description:Purpose: to observe differences in E. coli transcripts levels due to the absence of the tRNA modification queuosine and due to exposure to excess nickel. Methods: RNAseq was performed using the total RNA isolated from E. coli MG1655 WT and Δtgt strains grown in LB medium only or in LB added by 2 mM NiCl2. Results: in nickel-treated WT compared to untreated WT (WT_Ni/WT_LB) 734 genes were found up-regulated and 1,642 genes down-regulated in the treated cells; in the tgt mutant compared to the WT strain (tgt_LB/WT_LB), 417 genes were found up regulated and 1,053 genes down regulated in the tgt mutant. Conclusions: the gene expression pattern of the tgt deficient cell in LB medium resembles the one of the WT strain exposed to nickel, showing repression of iron-import genes and genes associated to anaerobic metabolism; increased resistance to nickel stress observed in the tgt mutant is explained by the repression of the nickel import system nikABCCDE.
2022-05-15 | GSE181239 | GEO
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.
2014-12-22 | GSE62679 | GEO
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.
2014-12-22 | GSE62678 | GEO
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.
2014-12-22 | GSE62677 | GEO