Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate UV-crosslinked SLBP RNP from cytoplasmic HeLa lysates. Mock immunoprecipitation were also performed and serve as a negative control.
Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate SLBP RNP from polyribosomal fractions of HeLa S3 lysates. Mock immunoprecipitation were also performed and serve as a negative control.
Project description:In neurons, mRNAs and associated RNA-binding proteins assemble into ribonucleoprotein (RNP) granules essential to regulate mRNA trafficking, local translation, and turnover. Dysregulation of RNA-protein condensation can disturb synaptic plasticity. We report that the novel lncRNA mimi is a constitutive and essential component of large cytoplasmic condensates (RNP granules) in fly neurons that are biochemically enriched by differential centrifugation. Here, employing relative iBAQ quantification we carry out a differential proteomic analysis of cytoplasmic RNP granules in wild-type versus delta-mimi mutant fly brains. Brain lysates from wild-type and mutant flies serve as a general proteome input control.
Project description:UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are the most frequently used methods to study protein-RNA interactions in the intact cells and tissues, but their relative advantages or inherent biases have not been evaluated. To benchmark CLIP and iCLIP method, we performed iCLIP with Nova protein, which is the most extensively studied protein by CLIP. Further, we assessed UV-C-induced cross-linking preferences, by exploiting the UV-independent formation of covalent RNA cross-links of the mutant RNA methylase NSUN2.
Project description:In the quick cross-linking ligation and sequencing of hybrids (qCLASH) protocol, RNA-protein (RNP) complexes of interest are UV cross-linked in living cells. The protein of interest is purified by immunoprecipitation (in this case, AGO bound to the miRNA and the target gene). The two interacting RNA molecules (e.g. miRNA-mRNA) are physically bound to each other by intermolecular RNA-RNA ligation, followed by library preparation and sequencing oh hybrids.
Project description:To investigate the possible consequences of APOBEC1 editing for miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins (Chi et al., 2009) was performed in BMDMs derived from wild-type and Apobec1-/- littermates.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is a viral RNA-binding protein essential for viral lytic gene expression. ORF57 binds to target RNA directly via interaction with cellular cofactors. To investigate the entire repertoire of ORF57-associated RNAs we performed UV cross-linking immunoprecipitatin (CLIP) experiment using an affinity-purified, highly specific anti-ORF57 antibody in KSHV-infected primariy effusion lymphoma BCBL-1 cells undegoing lytic virus replication.