Cell-specific RNA purification to study translatomes of mouse central nervous system

ABSTRACT: Summary Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types. For complete details on the use and execution of this protocol, please refer to Chai et al. (2017), Díaz-Castro et al. (2021), Díaz-Castro et al. (2019), Srinivasan et al. (2016), and Yu et al. (2018). Graphical abstract Highlights • A protocol for investigating cell-specific translatomes in mouse brain cells• Purification of RNA from genetically targeted cells using RiboTag tools• Recommended antibodies and oligonucleotides to assess brain-cell specificity• Sample quality assessment tips for generating the most reliable data Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types.

SUBMITTER: Bravo-Ferrer I 

PROVIDER: S-EPMC9127423 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature