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 (Van 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.
Our single-cell RNA-seq technology was further modified to create a simplified, streamlined workflow with very little hands-on time to address this need. We combined the reverse transcription (RT) and PCR amplification steps 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 and convenient while providing a comparable cDNA yield and the same unparalleled sensitivity and reproducibility as its predecessor.
Figure 1. Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows. The SMART-Seq v4 method (left) was modified to generate a simplified, streamlined workflow (SMART-Seq HT, right) with very little hands-on time. Once single cells have been obtained using FACS, the SMART-Seq HT Kit involves only three hands-on steps, while the original SMART-Seq v4 kit involves six hands-on steps. One key step in the SMART-Seq HT workflow is the one-step RT-PCR, performed using the One-Step RT-PCR Buffer, formulated specifically for optimal reverse transcription followed by efficient PCR cDNA amplification. The One-Step RT-PCR Buffer is directly compatible with AMPure bead purification without the need for the addition of Lysis Buffer. As with the original SMART-Seq v4 kit, the SMART-Seq HT Kit requires validation (quantification and assessment of high-molecular-weight, full-length cDNA) before the cDNA is used for sequencing library preparation.
