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Quantification of mRNA ribosomal engagement in human neurons using parallel translating ribosome affinity purification (TRAP) and RNA sequencing.

ABSTRACT: Translation regulation is a fundamental step in gene regulation with critical roles in neurodevelopment. Here, we describe three protocols to calculate the ribosomal-engagement levels of the transcriptome from in vitro-derived neuronal cells. The protocols described here include enrichment of in vitro-generated pluripotent-derived neurons, immunoaffinity purification of ribosome-bound RNAs, and calculation of the fraction of ribosome-engaged mRNAs. The ribosome-engaged RNA fraction is a measurement of the translation activity, and differences between genotype or growth conditions report change in translational regulation. For complete details on the use and execution of this protocol, please refer to Rodrigues et al. (2020).

SUBMITTER: Rodrigues DC

PROVIDER: S-EPMC7753199 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Quantification of mRNA ribosomal engagement in human neurons using parallel translating ribosome affinity purification (TRAP) and RNA sequencing.

Rodrigues Deivid Carvalho DC Mufteev Marat M Ellis James J

STAR protocols 20201218 1

Translation regulation is a fundamental step in gene regulation with critical roles in neurodevelopment. Here, we describe three protocols to calculate the ribosomal-engagement levels of the transcriptome from <i>in vitro</i>-derived neuronal cells. The protocols described here include enrichment of <i>in vitro</i>-generated pluripotent-derived neurons, immunoaffinity purification of ribosome-bound RNAs, and calculation of the fraction of ribosome-engaged mRNAs. The ribosome-engaged RNA fraction ...[more]

PMID: 33364619

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