Takara SMART-Seq mRNA HT LP and SMART-Seq mRNA HT

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

Data is shown for the SMART-Seq HT Kit (SS-HT) and SMART-Seq HT PLUS (SS-HT PLUS), which use the same chemistry as SMART-Seq mRNA HT and SMART-Seq mRNA HT LP.

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 Library Prep (SSlp) or the Nextera XT (Illumina) library prep kit, 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 SSlp are higher than those generated with Nextera XT. Panel C. SSlp identified 10% more genes than Nextera XT.

More Information

Applications

• cDNA synthesis and library preparation from single cells or total RNA for transcriptome sequencing
• Automation of next-generation sequencing library preparation

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.