Standard Operating Procedure for Next Generation Sequencing of RNA Isolated form Extracellular Vesicles within the Nanostructurome Methods Pipeline

The standard operating procedure (SOP) for next generation sequencing (NGS) of RNA from extracellular vesicles is presented. The SOP enables purification of small or total RNAs from isolated extracellular vesicles. Next, the SOP describes the sequencing of RNAs using NGS sequencing platform IonTorrent. In the presented protocol we define all the steps from isolation of RNA to sequencing data management, together with the cautions and checkpoints crucial for obtaining reliable high-quality sequencing reads, suitable for further analysis. NGS enables high-throughput DNA and RNA sequencing, offering accuracy, scalability, and cost-efficiency. RNA sequencing is widely used for analysing gene expression, alternative splicing, and non-coding RNAs. NGS also facil-itates the study of extracellular vesicles (EVs), which play a key role in intercellular communication, especially in plant-pathogen interactions. Insights gained could in-form novel disease management strategies, such as Spray-Induced Gene Silencing, for improved crop protection.