body fluids. EVs are highly heterogenous in size, ranging from 50 to 1000 nm in diameter. [1] Furthermore, they play key roles in the cell microenvironment and intercellular communication by transferring biological molecules including proteins, lipids, and nucleic acids. [2] Because of the relatively stable state in body fluids, EVs are recognized as potential noninvasive biomarkers in disease diagnostics and mediators in drug delivery. [3] EVs carry the characteristics of their parental cells; therefore, they are highly heterogeneous in molecular cargos. [4] Thus, EVs demonstrate a marked diversity, even in homogeneous or monoclonal cell populations. [5] The different molecular cargos in EVs are related to their function and effectiveness as disease biomarkers. Therefore, it is important to investigate the molecular cargos and their heterogeneity at the single-EV level.Traditional analytical methods to examine the molecular cargos of EVs, such as western blotting, polymerase chain reaction (PCR), and RNA-seq, usually rely on bulk assays to determine the overall molecular content from an entire EV population. The molecular cargo of an individual EV and the heterogeneity within an EV population is lost in bulk-level analyses. Recently, several new analytical methods, such as total internal reflection fluorescence microscopy (TIRFM), [6] digital PCR, [7] microarraybased chip, [8] and high-sensitivity flow cytometry combined with qPCR, [9] have been used to quantify a specific miRNA or mRNA in an single-EV or small identical subpopulations. However, a high-throughput analysis for the RNA profile of a single-EV is still lacking. Therefore, even though many studies have investigated the RNA cargos in bulk EVs, we still do not know how many genes or RNAs there are in a single-EV. This is a basic issue that needs to be addressed. Investigations of transcriptome for a single-EV are of great value to better define EV features, EV subpopulations, and clinical applications.The development of single-cell RNA sequencing (scRNA-seq) technologies, such as 10x Genomics Chromium, enable the detection of gene expression at the single-cell level, as well as the detection of cell heterogeneity and cell types. [10] It has been reported that EV populations are more heterogeneous than their parental cells. [11] Therefore, it is very important to build an experimental and analytical protocol to perform single-EV sequencing to reveal the transcriptome features of individual EVs.Although many studies have investigated functional molecules in extracellular vesicles (EVs), the exact number of ribonucleic acid molecules in a single-EV is unknown. Therefore, it is critical to explore the transcriptomic features and heterogeneity at the level of a single-EV. Here, using the 10x Genomics platform, the RNA cargos are profiled in single EVs derived from human K562 and mesenchymal stem cells. The key steps are labeling intact EVs using calcein-AM, detecting the EV concentration via flow cytometry, and using the CB2 algorithm with adaptive thres...
