The library input quantities (up to 310 µl of 2.5 nM pooled libraries) required to successfully load a high-throughput sequencer, such as the NovaSeq™ system, are much higher than the quantities (5 µl of 4 nM pooled libraries) required for a low-throughput sequencer like the NextSeq® system. For this reason, increased library yield is becoming a very desirable feature for library prep kits. The SMART-Seq v4 PLUS Kit (SSv4 PLUS kit) is designed to synthesize high-quality, full-length cDNA directly from multiple intact cells (up to 1,000 cells) or 10 pg–10 ng of total RNA, and generate a high yield of Illumina-ready sequencing libraries. The kit includes the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing for generating cDNA, the SMART-Seq Library Prep Kit (not sold separately) for fragmenting the cDNA and amplifying the library, and a unique dual index kit for allowing multiplexing of up to 96 samples (Figure 1, Panel A).
For cDNA generation, the SSv4 PLUS kit incorporates our patented SMART (Switching Mechanism at 5′ end of RNA Template) technology. This technology relies on the template-switching activity of reverse transcriptase to enrich for full-length cDNAs and to add defined PCR adapters directly to both ends of the first-strand cDNA (Chenchik et al. 1998). This ensures that the final cDNA libraries contain the entire length of the mRNA, including the 5' end. This strategy maintains a true representation of the original mRNA transcripts, which is a critical factor for gene expression analysis. SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, and allows direct cDNA synthesis from intact cells.
For library preparation from the generated full-length cDNA, the SSv4 PLUS kit also incorporates our patented library preparation technology. Unlike other library preparation kits, which are based on ligation of Y-adapters, our approach uses stem-loop adapters to construct high-quality libraries. The easy two-step workflow takes place in a single tube and is completed in about two hours. Sample mix-up, other handling errors, and sample loss are minimized since no intermediate purification steps or sample transfers are necessary (Figure 1, Panel B). The SMART-Seq v4 PLUS Kit also includes unique dual index (UDI) primers for amplification of indexed NGS-ready libraries, allowing the multiplexing of up to 96 samples. Once purified and quantified, the resulting libraries are ready for use on Illumina NGS instruments using standard Illumina sequencing reagents and protocols.