Subcellular RNA-seq for the Analysis of the Dendritic and Somatic Transcriptomes of Single Neurons

In neurons, local translation in dendritic and axonal compartments allows for the fast and ondemand modification of the local proteome. As the last few years have witnessed dramatic advancements in our appreciation of the brain's neuronal diversity, it is increasingly relevant to understand how local translation is regulated according to cell type. To this end, both sequencing-based and imaging-based techniques have recently been reported. Here, we present a subcellular single cell RNA sequencing protocol that allows molecular quantification from the soma and dendrites of single neurons, and which can be scaled up for the characterization of several hundreds to thousands of neurons. Somata and dendrites of cultured neurons are dissected using laser capture microdissection, followed by cell lysis to release mRNA content. Reverse transcription is then conducted using an indexed primer that allows the downstream pooling of samples. The pooled cDNA library is prepared for and sequenced in an Illumina platform. Finally, the data generated are processed and converted into a gene vs. cells digital expression table. This protocol provides detailed instructions for both wet lab and bioinformatic steps, as well as insights into controls, data analysis, interpretations, and ways to achieve robust and reproducible results. [GRAPHICS] .

Automated summary

In this study, a subcellular single cell RNA sequencing protocol is presented, which allows for molecular quantification from the soma and dendrites of single neurons and can be scaled up for the characterization of several hundreds to thousands of neurons. The protocol provides detailed instructions for both wet lab and bioinformatic steps, as well as insights into controls, data analysis, interpretations, and ways to achieve robust and reproducible results.