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SRP058313
Mus musculus
6 Downloadable Samples
Illumina HiSeq 2000
Description
We sequenced mRNA from FACS purified hair follicle bulge stem cells from 21 d old control and ILK-deficient mice, 3 biological replicates each Overall design: Examination of mRNA levels in control and ILK-deficient hair follicle bulge stem cells
Publication Title
Integrin-linked kinase regulates the niche of quiescent epidermal stem cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 48 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The glycopeptide antibiotic vancomycin (VCM) represents one of the last lines of defense against methicillin-resistant Staphylococcus aureus infections. However, vancomycin is nephrotoxic, but the mechanism of toxicity is still unclear.
Publication Title
Gene expression analysis reveals new possible mechanisms of vancomycin-induced nephrotoxicity and identifies gene markers candidates.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 25 Downloadable Samples
- IlluminaHiSeq2500, IlluminaHiSeq2000
Description
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We have developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing (Baltz and Munschauer et al. 2012). Our current work focuses on streamlining and extending protein occupancy profiling on poly(A)-RNA. Our objectives are to identify previously unknown protein-bound transcripts and, more importantly, to assess global and local differences in protein occupancy across different biological conditions. To this end, we have implemented poppi, the first pipeline for differential analysis of protein occupancy profiles. We have applied our analysis pipeline to pinpoint changes in occupancy profiles of MCF7 cells against already published HEK293 cells [GSE38157]. Overall design: We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled MCF7 cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced.
Publication Title
Differential protein occupancy profiling of the mRNA transcriptome.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2500
Description
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5'' to 3'' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3'' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5'' to 3'' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3'' UTRs. Overall design: Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were used to determine the protein-RNA interaction sites of RNA helicases MOV10 and UPF1 as well as MOV10 inactive variants using PAR-CLIP in combination with next generation sequencing. mRNA half-life changes of MOV10-targeted mRNA were determined by measuring mRNA half-lives by mRNA sequencing of mock and MOV10-depleted HEK293 cells.
Publication Title
MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.
Sample Metadata Fields
No sample metadata fields
View Samples- Sus scrofa
- 23 Downloadable Samples
- Affymetrix Porcine Genome Array (porcine)
Description
in vitro microarray study of transcriptional changes of jejunal cells
Publication Title
Deoxynivalenol Affects Cell Metabolism and Increases Protein Biosynthesis in Intestinal Porcine Epithelial Cells (IPEC-J2): DON Increases Protein Biosynthesis.
Sample Metadata Fields
No sample metadata fields
View Samples- Sus scrofa
- 14 Downloadable Samples
- Affymetrix Porcine Genome Array (porcine)
Description
in vitro microarray study of transcriptional changes of jejunal cells
Publication Title
Deoxynivalenol Affects Cell Metabolism and Increases Protein Biosynthesis in Intestinal Porcine Epithelial Cells (IPEC-J2): DON Increases Protein Biosynthesis.
Sample Metadata Fields
No sample metadata fields
View Samples- Caenorhabditis elegans
- 12 Downloadable Samples
- Illumina HiSeq 2000
Description
Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human Alu repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified new circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched compared to linear controls. By scoring the presence of reverse complementary sequences in human introns we predicted and experimentally validated novel circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyper-editing events. Knockdown of the double-strand RNA editing ADAR1 enzyme significantly and specifically up-regulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA:RNA interactions of introns form larger structures which promote circularization of embedded exons, while ADAR1 antagonizes circRNA expression by melting stems within these interactions. Thus, we assign a new function to ADAR1. Overall design: Examination of 12 samples in different stages of C.elegans development.
Publication Title
Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Murine Genome U74A Version 2 Array (mgu74av2)
Description
We used microarrays to identify genes differentially expressed between mouse RUNX2 -/- and wt embryonic humeri at stage E14.5
Publication Title
Detection of novel skeletogenesis target genes by comprehensive analysis of a Runx2(-/-) mouse model.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3'' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. Overall design: We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced
Publication Title
The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
Sample Metadata Fields
Treatment, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
- Illumina Genome Analyzer II
Description
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. Close to one third of these proteins, were neither previously annotated nor could be functionally predicted to bind RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3'' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of unexpected mRNA-binders with novel molecular functions participating in combinatorial post-transcriptional gene-expression networks. Overall design: To obtain a more detailed picture of the RNA present in the pooled precipitates of four consecutive oligo(dT)-purifications, we constructed a cDNA library by random priming of 4-thiouridine (4SU)- and 6-thioguanosine (6SG)-labeled RNA derived from UV-irradiated (365 nm)and non-irradiated cells. Digital gene expression analysis of the cDNA library of non-irradiated cells, labeled with 4SU and 6SG, was performed. To monitor the incorporation of photoreactive nucleotides into mRNA, we isolated 4SU- and 6SG-labeled RNA from the oligo(dT) precipitate of non-crosslinked cells by biotinylation and streptavidin purification (Dolken et al., 2008).
Publication Title
The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
Sample Metadata Fields
Treatment, Subject
View Samples