Full-Length Single-Cell RNA Sequencing with Smart-seq2

In the last few years single-cell RNA sequencing (scRNA-seq) has enabled the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types as well as the detailed analysis of the stochastic nature of gene expression. A large number of methods have been developed, varying in their throughput, sensitivity, and scalability. A major distinction is whether they profile only 5'- or 3'-terminal part of the transcripts or allow for the characterization of the entire length of the transcripts. Among the latter, Smart-seq2 is still considered the "gold standard" due to its sensitivity, precision, lower cost, scalability and for being easy to set up on automated platforms. In this chapter I describe how to efficiently generate sequencing-ready libraries, highlight common issues and pitfalls, and offer solutions for generating high-quality data.