Streamlined single-cell mRNA-seq with the SMART-Seq HT Kit
The core SMART-Seq HT Kit (SS-HT) and the SMART-Seq HT PLUS Kit (SS-HT PLUS) are automation-friendly kits that use oligo(dT) priming to generate high-quality, full-length cDNA directly from 1–100 cells or 10 pg–1 ng of total RNA. In addition to the core cDNA synthesis kit, the SS-HT PLUS kit also includes a library preparation kit and single-use unique dual index (UDI) plates to generate Illumina-compatible sequencing libraries, making it a complete end-to-end solution.
The core SMART-Seq HT Kit (SS-HT) and the SMART-Seq HT PLUS Kit (SS-HT PLUS) are automation-friendly kits that use oligo(dT) priming to generate high-quality, full-length cDNA directly from 1–100 cells or 10 pg–1 ng of total RNA. In addition to the core cDNA synthesis kit, the SS-HT PLUS kit also includes a library preparation kit and single-use unique dual index (UDI) plates to generate Illumina-compatible sequencing libraries, making it a complete end-to-end solution.
The SS-HT cDNA synthesis kit has a streamlined protocol optimized to work downstream of FACS and reduces hands-on time compared to SMART-Seq v4 kits, owing to the introduction of a convenient combined reverse transcription and PCR amplification step. The core SMART (Switching Mechanism at 5' End of RNA Template) technology powering the cDNA synthesis provides full-length transcript coverage, making it a powerful tool for studying gene expression in single cells, including splice junctions and alternative splicing. Oligo(dT) priming is used to specifically amplify mRNA from either high-quality total RNA (RIN >8) or intact cells. LNA technology improves the efficiency of template switching, which increases the number of genes identified compared to other methods.
The SS-HT PLUS kit incorporates our patented library preparation chemistry, which uses enzymatic fragmentation and stem-loop adapters to construct high-quality, Illumina-compatible libraries from the cDNA synthesized with the core kit. This two-step workflow takes place in a single tube and can be completed in about two hours—no intermediate purification steps are necessary.
If you prefer a random priming approach that will allow you to work with degraded samples and also includes library preparation and indexing reagents, we recommend our SMART-Seq Stranded Kit.
Overview
- Easily automate the streamlined workflow—process more samples in less time and maximize the space on your automation deck
- Unparalleled sensitivity—start with as little as one cell or 10 pg of total RNA (input range: 1–100 cells or 10 pg–1 ng of total RNA)
- High-quality RNA-seq data—capture full-length transcript coverage, a low percentage of rRNA reads, and a broad representation of GC-rich transcripts
- Single-tube workflows—complete cDNA synthesis and library preparation, in one tube each, for minimization of sample loss, sample mix-up, or other handling errors
- Increased sequencing power—include unique dual indexes to allow for the pooling of multiple samples and confident sequencing on the NovaSeq™ system
- High library yield—sequence on high-throughput sequencers with ease (such as the NovaSeq) or confidently save extra library for future runs
Interested in more data and FAQs about this product? Visit the NGS Learning Center.
SMART-Seq HT has a simplified workflow with less hands-on time
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 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 with the Illumina Nextera® XT DNA Library Preparation Kit.
SMART-Seq HT matches the performance of SMART-Seq v4
| Sequencing metrics comparing SMART-Seq v4 and SMART-Seq HT kits | |||||||
| RNA source | 10 pg Mouse Brain Total RNA | ||||||
| cDNA synthesis | SMART-Seq v4 | SMART-Seq HT | |||||
| Replicate | A | B | C | A | B | C | |
| Yield (ng) | 7.3 | 8.5 | 9.4 | 9.5 | 9.9 | 9.2 | |
| Number of transcripts | 14,168 | 13,934 | 14,147 | 14,510 | 14,650 | 14,553 | |
| 11,455 | 11,267 | 11,349 | 11,582 | 11,670 | 11,454 | ||
| Average Pearson/Spearman | 0.97/0.67 | 0.97/0.68 | |||||
| 0.96/0.66 | |||||||
| Proportion of reads mapped (%) | |||||||
| rRNA | 0.7 | 0.6 | 0.5 | 1.1 | 1.1 | 1.1 | |
| Mitochondria | 2.8 | 4.2 | 4.1 | 3.7 | 3.8 | 4.2 | |
| Genome | 92.3 | 90.8 | 86.6 | 89.1 | 89.0 | 87.8 | |
| Exons | 74.8 | 72.5 | 69.0 | 67.3 | 67.1 | 65.3 | |
| Introns | 13.3 | 14.1 | 13.3 | 16.9 | 16.9 | 17.4 | |
| Intergenic regions | 4.2 | 4.2 | 4.4 | 4.9 | 5.0 | 5.2 | |
Table 1. High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq HT kits. Libraries were prepared from 10 pg of Mouse Brain Total RNA. The output cDNA was converted into RNA-seq libraries using the Illumina Nextera XT DNA Library Preparation Kit and sequenced on an Illumina NextSeq® instrument (2 x 75 bp). Sequences were analyzed as described in the methods after normalizing all the samples to 13 million paired-end reads.
Figure 2. SMART-Seq HT PLUS generates more libraries and identifies more genes than Nextera XT. Panel A. SMART-Seq HT was used to produce cDNA from 10 pg of Mouse Brain Total RNA in triplicate. Illumina-compatible libraries were generated using SMART-Seq PLUS or Nextera XT (Illumina) library prep kits, and sequenced on a NextSeq 500 system. The reads were normalized to 4M paired-end reads and analyzed as described in the technical note. Panel B. Library yields obtained with SMART-Seq Library Prep Kit are higher than those generated with Nextera XT. Panel C. The SMART-Seq Library Prep Kit identified 10% more genes than Nextera XT.
More Information
Applications
- cDNA synthesis from single cells or total RNA for transcriptome sequencing
- cDNA outputs from the SS-HT kit can be used with the SMART-Seq Library Prep kit (not sold separately; sold as part of the SMART-Seq HT PLUS kit) for Illumina sequencing
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Product Components List to determine kit components. Certificates of Analysis and Product Components Lists are located under the Documents tab.
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