The emergence of next-generation sequencing (NGS) technology has encouraged intense interest in single-cell transcriptome analysis. Single-cell RNA-seq has been gaining prominence in basic and clinical research since it can be used to examine differential gene expression, alternative splicing, gene fusions, differentiation processes, tumor composition, and much more (Strijp et al. 2017; Burns, Brooks and Spencer 2016; Cheng et al. 2016; Lau et al. 2016). Single-cell RNA-seq overcomes problems inherent to bulk RNA sequencing, which averages gene expression over a cell population and thereby obscures the diversity of single-cell expression levels, which are susceptible to intrinsic stochastic variation and extrinsic variation from the microenvironment. Therefore, single-cell transcriptome information is critical for understanding fundamental biological phenomena and complex diseases (Kanter and Kalisky 2015).
Extracting meaningful biological information from the small amount of mRNA present in a single cell requires a library preparation method with exceptional sensitivity and reproducibility. By providing the capability to obtain full-length mRNA sequence information (as opposed to merely capturing transcript 3′ ends), the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (SMART-Seq v4) offers the most advanced single-cell RNA-seq method on the market (Fish et al. 2016; Sugiyama et al, 2016; Paul et al. 2017). However, this method is relatively low throughput and researchers may be interested in analyzing hundreds or thousands of individual cells per experiment.
To address this need, our single-cell RNA-seq technology was further modified to create a simplified, high-throughput workflow with very little hands-on time. The reverse transcription (RT) and PCR amplification steps were combined into a single RT-PCR step, so users can simply set up the one-step RT-PCR and walk away. The updated workflow, available in the SMART-Seq HT Kit, is extremely fast, convenient, and generates a comparable cDNA yield to its predecessor, all while providing the same unparalleled sensitivity and reproducibility.