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SRP003472
Danio rerio
2 Downloadable Samples
IlluminaGenomeAnalyzerII
Description
Precise 5' splice site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 snRNP specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C knockout mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. Surprisingly, genomewide RNA-Seq analysis of mutant versus wildtype embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.
Publication Title
RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation.
Sample Metadata Fields
No sample metadata fields
View Samples- Danio rerio
- 18 Downloadable Samples
Affymetrix Zebrafish Genome Array (zebrafish)
Description
The goal of the project was to isolate single miRNA-expressing cells labelled by GFP reporter genes under the control of endogenous miRNA promoters and analyze expression levels of miRNA target genes in these cells. GFP-positive miRNA-expressing cells and GFP-negative cells from the rest of the embryos were purified at the same developmental stage to the cellular resolution using fluorescent activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted
Publication Title
Coherent but overlapping expression of microRNAs and their targets during vertebrate development.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 4 Downloadable Samples
- Illumina Genome Analyzer IIx
Description
Small RNA deep sequencing analysis was conducted on primary human fibroblasts infected with human cytomegalovirus (HCMV). HCMV-encoded miRNAs accumulated to ~20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of novel HCMV miRNAs. Through crosslinking and immunoprecipitation of Argonaute-bound RNAs from infected cells, followed by high-throughput sequencing (Ago CLIP-seq), we obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Additionally, significant upregulation was observed during infection for a host miRNA cluster containing miR-96, miR-182 and miR-183. We also identified novel non-miRNA forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. Overall design: High-throughput profiling of smRNAs, Ago1-, and Ago2-associated miRNAs from HCMV-infected fibroblast cells. Wild-type HCMV Towne (Genbank FJ616285.1) was used for these studies.
Publication Title
High-resolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Homo sapiens
- 22 Downloadable Samples
- Illumina HiSeq 2500, NextSeq 550
Description
Transcriptional cofactors communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression, which is a hallmark of all multicellular organisms. However, the extent to which different cofactors display intrinsic specificity for distinct promoters is unclear. Testing intrinsic COF – core promoter (CP) compatibilities requires the systematic assessment of transcriptional activation for many CPs in the presence or absence of a given COF in an otherwise constant standardized reporter system. We therefore combined a plasmid-based high-throughput reporter assay, Self-Transcribing Active Core Promoter-sequencing (STAP-seq), with the specific recruitment of individual COFs to create a high-throughput activator bypass-like assay. Using this assay, we tested whether 5 different individually tethered human COFs (MED15, BRD4, EP300, MLL3 and EMSY) activate transcription from a selection of 12,000 candidate sequences encompassing different types of gene core promoters, enhancers and control sequences. In addition, we used the strong transcriptional activator P65 as a positive control and GFP as a negative control. We found that different COFs preferentially activate different CPs. For instance, MED15 prefers TATA-box containing CPs, while MLL3 preferentially activates CpG island promoters. The observed compatibilities between cofactors and promoters can explain how different enhancers specifically activate distinct sets of genes or alternative promoters within the same gene, and may underlie distinct transcriptional programs in human cells. Overall design: STAP-seq upon recruitment of individual transcriptional cofactor in HCT116 cells with 5 different cofactors and 2 controls, each in biological triplicate.
Publication Title
Transcriptional cofactors display specificity for distinct types of core promoters.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 21 Downloadable Samples
- Affymetrix Human Genome U133A Array (hgu133a)
Description
The purpose of the experiment was to generate a time course of gene expression following irradiation. The goal was then to model this data to extract hidden variables - chiefly, the activity profiles of the p53 transcription factor. Using this information the aim was to predict which transcripts changed by IR were targets of p53. Cells in log phase (1 x 106 ml-1) were ?-irradiated with 5 Gy at room temperature (RT) at a dose rate of 2.45 Gy per minute with a 137Cs ?-irradiator. Cells were harvested at 0, 2, 4, 6, 8, 10 and 12 hours, and RNA and protein were extracted (Trizol, Invitrogen). Affymetrix U133A arrays were hybridized as standard (www.affymetrix.co.uk). Array quality was determined using R and GCOS .rpt file values. The time course was replicated three times from independent cell preparations.
Publication Title
Ranked prediction of p53 targets using hidden variable dynamic modeling.
Sample Metadata Fields
Specimen part, Disease, Cell line, Time
View Samples- Homo sapiens
- 8 Downloadable Samples
- IlluminaHiSeq2500
Description
We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. Overall design: s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments
Publication Title
Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 8 Downloadable Samples
- Illumina HumanHT-12 V4.0 expression beadchip
Description
We used an Illumina Gene Expression Array to understand in more detail how T387 phosphorylation affects ISG induction. mRNA preparations from U6A cells expressing wild-type or T387A STAT2, treated with IFN- for 0, 4, 8, or 24 h, were analyzed.
Publication Title
Negative regulation of type I IFN signaling by phosphorylation of STAT2 on T387.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Rattus norvegicus
- 6 Downloadable Samples
- Illumina ratRef-12 v1.0 expression beadchip
Description
Hydroxyapatite-coated cellulose induces a quicker and stronger inflammatory response compared to uncoated cellulose. Furthermore, the coated cellulose increases the homing at circulating bone-marrow derived progenitor cells. For this reason, Illumina microarray was used to study the early gene expression of the forming granulation tissue in the hydroxyapatite-coated sponges.
Publication Title
Hemoglobin expression in rat experimental granulation tissue.
Sample Metadata Fields
Age, Specimen part, Time
View Samples- Homo sapiens
- 10 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
Basophil functional state was determined using the Flow2CAST Basophil Activation Test (BAT). Human basophils were isolated from whole blood of three house dust mite (HDM) responsive donors with reactive basophils and two donors with anergic basophils. Basophil isolation was performed using the EasySep Human Basophil Enrichment kit (Stemcell). Purity of the basophils was determined by flow cytometry and ranged from 96 - 99%. For stimulation, 75,000 basophils were incubated for 4 hr with or without 50 ul of anti-FCERI antibody (Bhlmann Laboratories) in a total volume of 200 ul of Stimulation Buffer provided by the Flow2CAST kit. After incubation, the basophils were collected and re-suspended in TRIzol Reagent (Life Technologies). Total RNA was extracted using double extraction protocol using the guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen), followed by a Qiagen RNeasy Micro clean-up procedure. cDNA and ST-ssDNA were prepared, fragmented and labeled according to Nugen WT Ovation Pico RNA Amplification and WT Ovation Exon kit and Encore Biotin protocols. The labeled ssDNA was hybridized on the Affymetrix Human GeneChip 1.0 ST Arrays, which subsequently were processed and stained using GeneChip Fluidics Station 450. The stained GeneChip Arrays were scanned in the Microarray Facility at the Biopolis Shared Facility using a GeneChip Scanner 3000. Quality control for the Arrays was performed using the Affymetrix Expression Console Software.
Publication Title
Systematic characterization of basophil anergy.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 33 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
35 Melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 microarrays were analysed for genes differentially expressed between cell lines carrying wild-type p14ARF and those with mutant 14ARF. All of these cell lines contained wild-type p53 (so that the effects of p14ARF mutations could be analysed without contamination from p53).
Publication Title
Gene expression profiling in melanoma identifies novel downstream effectors of p14ARF.
Sample Metadata Fields
No sample metadata fields
View Samples