Molecular characterization of neuronal cell types based on patterns of projection with Retro-TRAP

Nectow, Ekstrand and Friedman describe Retro-TRAP, an extension of the bacTRAP method, for projection-specific ribosome profiling of neurons. This two-component GFP-Nanobody system can, in theory, be adapted to analyze any GFP-expressing cell. Retro-TRAP (translating ribosome affinity purification) technology enables the synthesis of molecular and neuroanatomical information through the use of transgenic and viral approaches. In contrast to other methods that are used to profile neural circuits such as laser-capture microdissection and FACS, Retro-TRAP is a high-throughput methodology that requires minimal specialized instrumentation. Retro-TRAP uses an anti-GFP ribosomal tag (expressed virally or using transgenesis) to immunoprecipitate translating mRNAs from any population of neurons that express GFP. The protocol detailed here describes the rapid extraction of molecular information from neural circuits in mice using retrograde-tracing GFP-expressing viruses. This approach can be used to identify novel cell types, as well as to molecularly profile cell types for which Cre-driver lines are available, in defined presynaptic loci. The current protocol describes a method for extracting translating mRNA from any neural circuit accessible by stereotaxic injection and manual dissection, and it takes 2–4 weeks. Although it is not described here, this mRNA can then be used in downstream processing applications such as quantitative PCR (qPCR) and high-throughput RNA sequencing to obtain 'molecular connectomic' information.