SMART-Seq mRNA HT LP and SMART-Seq mRNA HT
NOTE: SMART-Seq mRNA HT LP and SMART-Seq mRNA HT are equivalent replacements for the SMART-Seq HT PLUS Kit and SMART-Seq HT Kit (see a complete list of kits with new names and the existing kits they will replace here).
The updates (listed below) do not in any way impact the protocols or functional performance of these kits.
- New kit sizes match those of Unique Dual Index kits (24 rxns, 96 rxns, 384 rxns)
- For added flexibility indexes are not included in the library prep (LP) kit; a choice of indexing primers are sold separately (see Unique Dual Index kits)
NOTE: SMART-Seq mRNA HT LP and SMART-Seq mRNA HT are equivalent replacements for the SMART-Seq HT PLUS Kit and SMART-Seq HT Kit (see a complete list of kits with new names and the existing kits they will replace here).
The updates (listed below) do not in any way impact the protocols or functional performance of these kits.
- New kit sizes match those of Unique Dual Index kits (24 rxns, 96 rxns, 384 rxns)
- For added flexibility indexes are not included in the library prep (LP) kit; a choice of indexing primers are sold separately (see Unique Dual Index kits)
SMART-Seq mRNA HT and SMART-Seq mRNA HT LP 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. While SMART-Seq mRNA HT contains the core cDNA synthesis kit, SMART-Seq mRNA HT LP also includes a library preparation kit. Up to 384 multiplex Illumina-ready sequencing libraries can be generated using SMART-Seq mRNA HT LP with Unique Dual Index kits (sold separately).
SMART-Seq mRNA HT has a streamlined protocol optimized to work downstream of FACS and reduces hands-on time, 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.
SMART-Seq mRNA HT LP, which incorporates our patented library preparation chemistry, uses enzymatic fragmentation and stem-loop adapters to construct high-quality, Illumina-compatible libraries. 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
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.
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