Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022:2537:37-49.
doi: 10.1007/978-1-0716-2521-7_3.

An Optimized Protocol for the Mapping of Cell Type-Specific Ribosome-Associated Transcript Isoforms from Small Mouse Brain Regions

Giulia Di Bartolomei  1 , Peter Scheiffele  2
Affiliations

An Optimized Protocol for the Mapping of Cell Type-Specific Ribosome-Associated Transcript Isoforms from Small Mouse Brain Regions

Giulia Di Bartolomei et al. Methods Mol Biol. 2022.

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.

Keywords: Alternative Splicing; Cell-type specificity; RiboTag; Ribosome-affinity purification; TRAP; Transcriptomics; mRNA isolation.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

References

    1. Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F, van der Zwan J, Haring M, Braun E, Borm LE, La Manno G, Codeluppi S, Furlan A, Lee K, Skene N, Harris KD, Hjerling-Leffler J, Arenas E, Ernfors P, Marklund U, Linnarsson S (2018) Molecular architecture of the mouse nervous system. Cell 174(4):999–1014 e1022 - DOI
    1. Zeng H, Sanes JR (2017) Neuronal cell-type classification: challenges, opportunities and the path forward. Nat Rev Neurosci 18(9):530–546 - DOI
    1. Nguyen TM, Schreiner D, Xiao L, Traunmuller L, Bornmann C, Scheiffele P (2016) An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons in the mouse hippocampus. eLife 5:e22757 - DOI
    1. Wamsley B, Jaglin XH, Favuzzi E, Quattrocolo G, Nigro MJ, Yusuf N, Khodadadi-Jamayran A, Rudy B, Fishell G (2018) Rbfox1 mediates cell-type-specific splicing in cortical interneurons. Neuron 100(4):846–859.e7 - DOI
    1. Gonatopoulos-Pournatzis T, Niibori R, Salter EW, Weatheritt RJ, Tsang B, Farhangmehr S, Liang X, Braunschweig U, Roth J, Zhang S, Henderson T, Sharma E, Quesnel-Vallieres M, Permanyer J, Maier S, Georgiou J, Irimia M, Sonenberg N, Forman-Kay JD, Gingras AC, Collingridge GL, Woodin MA, Cordes SP, Blencowe BJ (2020) Autism-Misregulated eIF4G microexons control synaptic translation and higher order cognitive functions. Mol Cell 77(6):1176–1192 e1116 - DOI

Publication types

LinkOut - more resources