The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5’cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5’cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP. Overall design: We used two biological replicates of S. cerevisiae that was grown to exponential phase (OD600 ~1) in rich media (YPAD). Samples where processed until the dephosphorylation step (CIP treatment). After that each sample was split in 4 aliquots: TAP treatment, Cap-Clip treatment, Decapping Pyrophosphohydrolase treatment or no treatment (negative control). From that step all samples are processed in parallel.
Widespread Co-translational RNA Decay Reveals Ribosome Dynamics.
Treatment, Subject
View SamplesThe use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae. Overall design: Experimental benchmark of five different protocols (3tfill, bpmI, internal, rnaseq and yoon) for genome-wide identification of polyadenylation sites in Saccharomyces cerevisiae and transcript quantification. RNA was extracted from WT cells grown in glucose (ypd) or galactose (ypgal) as carbon source. The same RNA was used for 3 independent library constructions (technical replicates, rep).
An efficient method for genome-wide polyadenylation site mapping and RNA quantification.
Cell line, Subject
View SamplesHere we quantified the transcription start site usage in a WT strain (BY4741) and a ?set2 strain associated with the appearence of cryptic transcription start sites. Overall design: Transcription start site usage was quantified using the 5’cap sequencing aproach for S. cerevisiae strains. Biological duplicates were included.
A high-throughput ChIP-Seq for large-scale chromatin studies.
Cell line, Subject
View SamplesSequencing of 5' and 3'ends and RNA-seq of PROMPT and mRNA molecules from control and exosome-depleted cells. Overall design: CAGE, 3'TAG and RNAseq library construction from RNA extracted from control and exosome-depleted cells.
Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters.
Specimen part, Cell line, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors.
Specimen part
View SamplesRecent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally-derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally-derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from facial (neural crest-derived) and foreskin (mesodermally-derived) dermis, and the mesodermally-derived SKPs can make myelinating Schwann cells. Thus, non-neural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally-defined lineage boundaries are more flexible than widely thought.
Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors.
Specimen part
View SamplesRecent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally-derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally-derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from facial (neural crest-derived) and foreskin (mesodermally-derived) dermis, and the mesodermally-derived SKPs can make myelinating Schwann cells. Thus, non-neural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally-defined lineage boundaries are more flexible than widely thought.
Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors.
Specimen part
View SamplesThe role of androgen in breast cancer development is not fully understood although androgen receptors (AR) have been identified in breast cancer clinical samples and cell lines. However the whole spectra of androgen actions cannot be accounted to the classic AR mode of action and the possible existence of a cell surface AR has been suggested. Indeed androgens like all steroids have been reported to trigger membrane initiated signaling activity and exert specific actions. Androgens acting on the membrane can rapidly activate kinase signaling pathways and ultimately could affect gene expression. However, the molecular nature of membrane androgen binding sites represents another major persisting question. In the present study, we investigated early transcriptional effects of testosterone and the impermeable testosterone-BSA conjugate, in two breast cancer cell lines, in an attempt to decipher specific genes modified in each case, providing evidences about specific membrane initiating actions. Our data indicate that the two agents tested affect the expression of several genes. A group of genes were commonly affected while others were uniquely modified by each agent. In MDA-MB-231 cells, that are AR negative, the majority of genes affected by testosterone were also affected by testosterone-BSA indicating a membrane action. Subsequent analysis revealed that the two agents trigger different molecular pathways and cellular/molecular functions, suggestive of a molecular heterogeneity of membrane and intracellular AR. In addition, the phenotypic interactions of membrane-acting androgen with growth factor were verified at the transcriptomic level. Finally an interesting interplay between membrane-acting androgen with inflammation-related molecules, with potential clinical implications was revealed.
Conjugated and non-conjugated androgens differentially modulate specific early gene transcription in breast cancer in a cell-specific manner.
Specimen part, Cell line
View SamplesTransgenic rice plants expressing isopentenyltransferase (IPT), an enzyme that catalyzes the rate-limiting step in CK synthesis under the control of SARK, a maturation- and stress-inducible promoter. Increased CK production resulted in sink source alteration and enhanced drought tolerance of the transgenic plants.
Cytokinin-mediated source/sink modifications improve drought tolerance and increase grain yield in rice under water-stress.
Age, Specimen part
View SamplesKeratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin.
Keratinocyte growth factor induces gene expression signature associated with suppression of malignant phenotype of cutaneous squamous carcinoma cells.
Specimen part, Disease
View Samples