Neuronal synapses contain rich repertoires of RNAs that can be used for onsite translation and modulation of their proteomes. Here, we present a protocol to recover a synaptosome-enriched fraction adapted to presynaptic RNA profiling, starting from adult Drosophila heads. We describe steps for generating synaptosomes through gentle lysis, followed by centrifugation and a density-based sucrose gradient. We then detail the procedures for validation of the protocol. This protocol can be applied to frozen material. For complete details on the use and execution of this protocol, please refer to de Queiroz et al. 1 • Protocol to profile presynaptic RNA content in adult Drosophila brain • Optimized head lysis and preservation of synaptosome RNA integrity • Density-based fractionation for synaptosome isolation • Provides total RNA material that can be used for NGS sequencing or RT-qPCR Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Neuronal synapses contain rich repertoires of RNAs that can be used for onsite translation and modulation of their proteomes. Here, we present a protocol to recover a synaptosome-enriched fraction adapted to presynaptic RNA profiling, starting from adult Drosophila heads. We describe steps for generating synaptosomes through gentle lysis, followed by centrifugation and a density-based sucrose gradient. We then detail the procedures for validation of the protocol. This protocol can be applied to frozen material.
Blot et al. (2026) studied this question.