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.
Differential protein occupancy profiling of the mRNA transcriptome.
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View SamplesHuman embryonic stem cells were differentiated into peripheral sensory neurons via the intermediate generation of neural crest like cell (NCC). Using various markers we identified these cells as LTMR. We then analyzed there complete transcriptional profile in comparison to the intermediate neural crest like cells. Overall design: mRNA expression data of human ESC-derived sensory neuron clusters (10-20 cells) and human ESC-derived neural crest like cells (~100 cells) was generated by illumina deep sequencing
PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.
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View SamplesDermal lymphatics form a network that connects all the hair follicles in skin and localize in proximity to the Hair Follicle Stem Cell. RNA sequencing analyses of isolated dermal lymphatics at two different time points of the hair follicle cycle (P55 and P70) indicate the existence of dynamic signaling networks associated with lymphatic remodeling, immune trafficking, and HF signaling. Overall design: Prox1CreERT2; ROSA26-LSL-eYFP mice of P55 (Mid Telogen) and P70 (Late telogen) were sacrificed and eYFP positive cells were isolated from the backskin.
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo.
Treatment, Subject
View SamplesCXCL5, a strong neutrophil-chemoattractant, has been reportet to be expressed in different cancer entities with diverse outcomes in disease progression. Contradictory outcome in disease progression in different tumor entities might be explained by a tumor type specific expression pattern of chemokines, chemokine receptors and growth factors that act in concert with CXCL5. This study evaluates the impact of CXCL5 expression on the tumor mircoenvironment in a syngeneic mouse melanoma model. Overall design: 105 B16F1 and B16F1-CXCL5 murine melanoma were injected intradermally into the flank skin of C57BL/6 J mice. Primary tumors were grown up to 250-350mm³, excised, snap frozen and then processed for RNA sequencing.
CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.
Specimen part, Treatment, Subject
View SamplesTranscriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has informed our understanding of blood differentiation and leukemogenesis, but a similarly transformative analysis of the embryonic origins of hematopoiesis is lacking. To address this issue, we acquired gene expression profiles of developing HSC purified from over 2500 dissected murine embryos and adult mice, and applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs emerging from hemogenic endothelium, and definitive HSCs. We functionally validated several candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos, confirming changes in the expression of HSC markers runx1 and c-myb in the aorta-gonads-mesonephros (AGM), the site of definitive HSC specification. Moreover, we found that HSCs derived from differentiating embryonic stem cells in vitro (ESC-HSC) most closely resemble definitive HSC, yet lack a signature indicative of specification by Notch signaling, which likely accounts for their deficient lymphoid development. Our analysis and accompanying web resource will accelerate the characterization of regulators of HSC ontogeny, facilitate efforts to direct hematopoietic differentiation and cell fate conversion, and serve as a model to study the origins of other adult stem cells.
The transcriptional landscape of hematopoietic stem cell ontogeny.
Specimen part
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.
Specimen part, Cell line
View SamplesAnalysis of CD41 single positive, VE-cadherin single positive, double positive, and double negatvie populations among 7AAD-CD45- cells from day 6 EBs
Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition.
Specimen part
View SamplesDNA methylation is often inversely correlated with gene expression.
Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.
Cell line
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells.
Specimen part, Treatment
View SamplesGene expression profiles of human embryonic stem cells, fibroblasts, and fibroblast-derived induced pluripotent stem cells
Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells.
Specimen part
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