Smartseq

SMART-seq, short for Switching Mechanism at the 5' end of RNA Transcript, is a method for generating full-length cDNA libraries from small amounts of RNA, notably from single cells. It enables transcriptional profiling with near full-length transcripts, facilitating detection of splice isoforms and other transcript features. The protocol begins with reverse transcription using an oligo-dT primer to capture mRNA, followed by template-switching at the 5' end of the cDNA to add a universal priming site. This enables subsequent PCR amplification of the full-length cDNAs, which are then prepared into sequencing libraries compatible with high-throughput platforms such as Illumina.

Variants of SMART-seq include SMART-seq2, an improved version that increases sensitivity and uniformity of transcript coverage, reducing dropout events. Further iterations have adapted the approach for ultra-low input or single-cell workflows and, in some cases, incorporated unique molecular identifiers to improve transcript counting and quantify molecules more accurately.

Applications of SMART-seq and its variants span single-cell transcriptomics, developmental biology, neuroscience, and cancer research, where full-length transcript information aids analyses of alternative splicing, isoform expression, and allele-specific expression. Advantages include high sensitivity and the ability to recover full-length transcripts; limitations include residual amplification bias, higher cost and labor relative to some droplet-based single-cell methods, and a reliance on high-quality RNA. Overall, SMART-seq remains a foundational approach for deep, full-length transcript profiling in low-input samples.