Abstract
Over the past years, technological advances in transcriptomics provided deep insights into gene expression programs and their role in tissue organization and cellular functions. The isolation of ribosome-associated transcripts is a powerful approach for deep profiling of cell type–specific transcripts, and particularly well-suited for quantitative analysis of transcript isoforms. This method employs conditional ribosome epitope-tagging in genetically defined cell types, followed by affinity-isolation of ribosome-associated mRNAs. Advantages of this approach are twofold: first, the method enables rapid retrieval of mRNAs without tissue dissociation and cell sorting steps. Second, capturing of ribosome-associated mRNAs, enriches for transcripts recruited for active translation, therefore providing an approximation to the cellular translatome. Here, we describe one application of this method for the identification of the transcriptome of excitatory neuronal cells (mitral and tufted cells) of the mouse olfactory bulb, through RiboTag isolation from the vGlut2-IRES-cre mouse line as genetic driver of endogenously tagged ribosome expression.
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Acknowledgments
We are thankful to O. Mauger and E. Furlanis for constructive comments on the chapter manuscript and to T.M. Nguyen, L. Traunmüller, E. Furlanis, and S. Falkner for discussions and help in setting up the protocol for RiboTRAP purifications. Work in the laboratory was supported by funds to P.S. from the Swiss National Science Foundation, a European Research Council Advanced Grant (SPLICECODE), EU-AIMS and AIMS-2-TRIALS which are supported by the Innovative Medicines Initiatives from the European Commission. The results leading to this publication has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement no. 777394. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and AUTISM SPEAKS, Autistica, SFARI. The Scheiffele Laboratory is an associate member of the Swiss National Science Foundation’s National Competence Centre for Research (NCCR) RNA and Disease.
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Di Bartolomei, G., Scheiffele, P. (2022). An Optimized Protocol for the Mapping of Cell Type–Specific Ribosome-Associated Transcript Isoforms from Small Mouse Brain Regions. In: Scheiffele, P., Mauger, O. (eds) Alternative Splicing. Methods in Molecular Biology, vol 2537. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2521-7_3
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DOI: https://doi.org/10.1007/978-1-0716-2521-7_3
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