Project description:The easyCLIP protocol describes a method for both normal CLIP library construction and the absolute quantification of RNA cross-linking rates, data which could be usefully combined to analyze RNA-protein interactions. Using these cross-linking metrics, significant interactions could be defined relative to a set of random non-RBPs. The original easyCLIP protocol did not use index reads, required custom sequencing primers, and did not have an easily reproducible analysis workflow. This short paper attempts to amend these deficiencies. It also includes some additional technical experiments and investigates the usage of alternative adapters. The results here are intended to allow more options to easily perform and analyze easyCLIP.
Project description:DNA methylation (5mC) plays important roles in epigenetic regulation of genome function, and recently the TET1-3 hydroxylases have been found to oxidize 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC), and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins with preferential binding to 5-methylcytosine (5mC) and its oxidized forms where readers for 5mC and 5hmC (5-hydroxymethylcytosine) showed little overlap while further oxidation forms enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine containing DNA using mouse embryonic stem cell (mESCs) extracts. The dataset contains 3 biological replicates each of mouse ES cell nuclear proteins binding to Pax6 and FGF15 promoter sequences containing different modified forms of cytosine. Data analysis: Mass spectrometric data were processed using Proteome Discoverer v1.3 and searched against a mammalian entries in Uniprot 2011.09 using Mascot v2.3 with the following parameters: Enzyme - trypsin; max 1 missed cleavage; Precursor Mass Tolerance - 10 ppm; Fragment Mass Tolerance - 0.6 Da; Dynamic Modification - Oxidation (M); Static Modification - Carbamidomethyl at C.
Project description:Omics Analysis of Pancreatic Cancer Platelets Reveals Active “Education”, including Dysregulation of Splicing Signature and Inhibition of SPARC by Modified Forms of miRNAs
Project description:Bloodstream forms of T brucei s427 2T1 T7 p’5neo5’-ESAG9 3’UTR (long form) bloodstream forms were grown in vitro. They contained the NeoR gene under the control of the ESAG9 gene 3'UTR. Selection was based on increased resistance to G418 through the silencing of an ESAG9 repressor, leading to increased neoR expression
Project description:Fibroblast Growth Factor-2 (FGF-2) induces cell proliferation, cell migration, embryonic development, cell differentiation, angiogenesis and malignant transformation. The four forms of FGF-2 (Low Molecular Weight) and (High Molecular Weights) are alternative translation products, and have a different subcellular localization: the high molecular weight (HMWFGF-2) forms are nuclear while the low molecular weight form, (LMWFGF-2) is mainly cytoplasmic. Our previous work demonstrated NIH 3T3 cells expressing different FGF-2 forms, displayed a different phenotype, suggesting that nuclear and cytoplasmic forms of FGF-2 may have different functions. Here we report a cDNA microarray-based study in NIH 3T3 fibroblasts expressing different FGF-2 forms. Several candidate genes that affect cell-cycle, tumor suppression, adhesion and transcription were identified as possible mediators of the HMWFGF-2 phenotype and signaling pattern. These results demonstrated that HMWFGF-2 and LMWFGF-2 target the expression of different genes. Particularly, our data suggest that HMWFGF-2 forms may function as inducers of growth inhibition and tumor suppression activities. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:We have developed a modified iteration of a chromosome conformation capture (V3C-T5-seq) assay to degrade non-cccDNA forms of HBV to identify where cccDNA forms of the HBV genome localizes.
