Project description:We report mRNA profiles of subcellularly localized transcriptomes (soma and neurite) of two mouse cell lines, N2A and CAD, as well as primary cortical neurons from E18.5 mice. We also performed this fractionation and sequencing after RNAi knockdown (cell lines) or in knockout mice (primary cortical neurons) of the RNA-binding proteins muscleblind 1 and 2 (Mbnl1 and Mbnl2). Fractionate neurons using porous transwell membranes. Isolate poly-A RNA.
Project description:We performed SlamSeq (thiol(SH)-linked alkylation for metabolic sequencing) to estimate mRNA half-lives in subcellular compartments (neurites, soma-cytoplasm and nucleus) of primary cortical neurons.
Project description:To study the effect of Larp1 on the abundance and subcellular localization of 5'TOP containing mRNAs, Larp1 was depleted from mouse primary cortical neurons using shRNAs. RNA from subcellular compartments (neurite and soma cytoplasm) was isolated and sequenced in parallel with scrambled control shRNA expressing samples.
Project description:To study the correlation between mRNA stability and subcellular mRNA localisation we globally interferred with mRNA degradation in primary cortical neurons by overexpressing a catalytic mutant of deadenylase CAF1 that functions as a dominant negative form. Neurons were separated into subcellular compartments (neurite, soma-cytoplasm and nucleus) and sequenced in parallel with respective compartments from GFP- expressing control neuons.
Project description:For identification of transcripts enriched in neurites of primary cortical neurons, the cells were plated on a microporous membrane for isolation of neurites and soma. Extracted RNA was used for preparation of mRNA-seq, total RNA-seq or smRNA-seq libraries and Illumina sequencing. For neuronal zipcode identification protocol (N-zip) in mouse cortical neurons, we combined a massively parallel reporter assay with neurites/soma separation. Neurons, grown on a microporous membrane, were infected with a library of around 5000 oligos tiled across 3'UTRs of selected neurite-enriched transcripts, cloned downstream of GFP coding sequence. RNA was extracted from soma and neurites and reverse transcribed into cDNA. Amplicon libraries of 3'UTR reporters were prepared and subjected to Illumina sequencing. In the second round of N-zip, a library containing selected reporters from the first N-zip and their mutagenized versions (around 6000 oligos) were used. In the following rounds, N-zip was combined with knockdown of selected genes.
Project description:To study how the codon composition of the coding sequence affects subcellular localization of mRNA in an unbiased way, mouse primary cortical neurons were transfected with a collection of human open reading frames (ORFs) with identical untranslated regions, separated into soma and neurites, and subjected to RNA sequencing with specific amplification of the ORFs.
Project description:We have perturbed mRNA degradation machinery and investigated the change in subcellular localization of mRNA in mouse primary cortical neurons (mPCNs). Mutant mPCN line harbouring a ponasteroneA-inducible heterozygous dominant-negative Caf1 (dnCaf1) was generated, separated into neuronal compartments (soma and neurites) and sequenced in parallel with compartments from the GFP-transfected cells (GFP, negative control).
Project description:We have perturbed mRNA degradation machinery and investigated the change in subcellular localization of mRNA in Ascl1-induced neurons (iNeurons). Mutant iNeuron line harbouring a ponasteroneA-inducible heterozygous dominant-negative Caf1 (dnCaf1) was generated, separated into neuronal compartments (soma and neurites) and sequenced in parallel with compartments from the Wild Type iNeurons (WT). Differential localization of mRNA between soma and neurites in the WT was then compared to the localization in dnCaf1 in order to identify the changes in localization.
Project description:We have perturbed RNA methylation machinery and investigated the change in subcellular localization of mRNA in Ascl1-induced neurons (iNeurons). Mutant iNeuron line harbouring tagged endogenous Mettl3 was generated which allowed for the selective and targeted depletion of Mettl3 protein with the addition of dTAG. Cells were treated with dTAG for 40 hours then separated into compartments (neurites and soma) and then sequenced in parallel with samples which received no dTAG treatment.
Project description:To assess neuronal expression divergence between mice and rats, we used the Affymetrix array platform to assay the transcriptomes of micro-dissected individual soma and pool of dendrites of hippocampal neurons in dispersed primary cell cultures from rat and mouse. Using microdissected soma and dendrites from primary cultures of hippocampal neurons of two mouse strains (C57BL/6 and Balb/c) and one rat strain (Sprague-Dawley), we investigate via microarrays, subcellular localization of mRNAs in neurons