Methods for global gene expression analysis in individual cells have important applications in biology and medicine. This webinar describes the Smart-Seq protocol for polyA+ RNA analysis, applicable at single-cell levels. Compared to existing methods, the Smart-Seq protocol has improved read coverage across transcripts, significantly enhancing analysis of alternative transcript isoforms and improving SNP identification. The sensitivity and quantitative accuracy of the Smart-Seq method for single-cell transcriptomics have been evaluated with total RNA dilution and spike-in RNAs. This method has been proven using cancer cell lines, circulating tumor cells from melanomas, and early embryonic cells. The Smart-Seq protocol provides a robust method for addressing fundamental biological questions that require transcriptome profiling in rare or single cells.