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SRP066351
Gallus gallus
4 Downloadable Samples
Illumina HiSeq 2000
Description
We employ RNA-seq of FACS sorted cell populations to identify genes that are enriched in cranial neural crest in relationship to the trunk. Overall design: Transcriptional profiling of delaminating cranial and trunk neural crest subpopulations.
Publication Title
Reprogramming of avian neural crest axial identity and cell fate.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Proprioception relies on two main classes of proprioceptive sensory neurons (pSNs). These neurons innervate two distinct peripheral receptors in muscle, muscle spindles (MSs) or Golgi tendon organs (GTOs), and synapse onto different sets of spinal targets, but the molecular basis of their distinct pSN subtype identity remains unknown.
Publication Title
The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors.
Sample Metadata Fields
Sex, Specimen part
View Samples- Homo sapiens
- 10 Downloadable Samples
- Illumina HiSeq 2500
Description
RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report expansion of TimeLapse sequencing (TimeLapse-seq) to the guanosine analogue, 6-thioguanosine (s6G), which can be recoded under RNA-friendly nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics. Overall design: Distinguishing newly made from preexising RNA using RNA-sequencing of 6-thioguanosine containing RNA, which was subjected to TimeLapse chemistry to induce G to A mutations in newly-made RNA.
Publication Title
Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-Thioguanosine.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Saccharomyces cerevisiae
- 33 Downloadable Samples
- Illumina HiSeq 2000
Description
During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a “checkpoint” during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo. Overall design: Total RNA was collected from wild-type and analog-sensitive Kin28 strains treated with reversible inhibitor 1-NAPP-1, irreversible inhibitor CMK, and solvent control DMSO. Equivalent ratios of S. pombe : S. cerevisiae cells were added to each sample before RNA extraction for normalization of read counts after sequencing. Nascent RNA was purified from total RNA by 4-thiouracil labeling, biotinylation, and streptavidin-pulldown. As a negative control, nascent RNA was also extracted from total RNA from cells that had not been treated with 4-thiouracil.
Publication Title
Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Mus musculus
- 16 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Dicer deficiency reveals microRNAs predicted to control gene expression in the developing adrenal cortex.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 16 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
MicroRNAs (miRNAs) are small, endogenous, non-protein coding RNAs that are an important means of post-transcriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer KO adrenals demonstrated a significant loss of Sf1 expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by anti-PCNA staining. To further characterize the embryonic adrenals from Dicer KO mice, we performed microarray analyses for both gene expression and miRNA on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21 that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer KO adrenals. Together these data suggest a role for miRNA mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.
Publication Title
Dicer deficiency reveals microRNAs predicted to control gene expression in the developing adrenal cortex.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens, Rattus norvegicus
- 1 Downloadable Sample
- Illumina HumanHT-12 V4.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 1 Downloadable Sample
- Illumina HumanHT-12 V4.0 expression beadchip
Description
Objective: The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDHhigh) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDHhigh cells in ovarian cancer. Methods: We compared ALDHhigh to ALDHlow cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. Results: ALDHhigh cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDHhigh cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following drugable targets were consistently expressed in the ALDHhigh cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18 / FGFR3. Conclusions: Based on functional characterization, ALDHhigh ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified drugable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes.
Publication Title
Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 18 Downloadable Samples
- Affymetrix Human Exon 1.0 ST Array [probe set (exon) version (huex10st)
Description
Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome-depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately transcriptional dysregulation.
Publication Title
Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription.
Sample Metadata Fields
Cell line
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
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