Single cell profiling of total RNA using Smart-seq-total
We describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts only. Overall design: We sequenced total RNA from individual human primary dermal fibroblasts, HEK293T and MCF7 cells sorted in 384-well plates and processed in 1/10 of the standard Smart-seq2 volume. In parallel, we analyzed the RNAome of primed pluripotent stem cells and that of individual cells obtained from dissociated embryoid bodies at days 4, 8 and 12 of culture.