SMART-Seq HT Kit
SMART-Seq HT Kit
Brand: Takara Bio.
In stock
SKU
SMART-Seq HT Kit
Designed for the synthesis of high-quality cDNA directly from 1–100 intact cells or ultra-low amounts of total RNA (10–1,000 pg)
Streamlined single-cell mRNA-seq with the SMART-Seq HT Kit
The SMART-Seq HT Kit (SS-HT) is an automation-friendly kit that uses oligo(dT) priming to generate high-quality, full-length cDNA directly from 1–100 cells or 10 pg–1 ng of total RNA.
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 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
- Increased sequencing power—include unique dual indexes to allow for the pooling of multiple samples and confident sequencing on the NovaSeq™ system
Application
- 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
Gene expression data obtained from FACS-sorted 293T cells
High reproducibility of gene expression data obtained from FACS-sorted 293T cells using the SMART-seq v4 and SMART-Seq HT kits. Libraries generated from twenty-one individual 293T cells (table, top panel) were further analyzed to evaluate the reproducibility of gene expression measurements obtained for each cell with the SMART-Seq v4 kit (SSv4_1 to SSv4_12) and the SMART-Seq HT Kit (HT_1 to HT_9)(lower panel). The hierarchical clustering heat map shows Euclidean distances between all the cells and reports Pearson correlations ranging from 0.74 to 0.97. While the best correlations are observed between cells prepared with one or the other kit, the correlations are still very high between the two kits and the cells are not clustering based on the library preparation method. These data demonstrate that the modified workflow in the SMART-Seq HT Kit does not introduce major bias in measurement of gene expression levels.
Comparison of expression level by gene GC content between the SMART-Seq v4 and SMART-Seq HT kits
Comparison of expression level by gene GC content between the SMART-Seq v4 and SMART-Seq HT kits. The libraries made from 10 pg of Mouse Brain Total RNA shown in the table (Figure 2) were further analyzed for GC content representation (see table, top panel). Genes were binned by GC content, and correlation plots were used to visualize the reproducibility of the expression levels (FPKM) of genes in each bin. The average gene counts are very reproducible for replicate samples analyzed using the SMART-Seq v4 (Panel A) or SMART-Seq HT kits (Panel B). Genes with high or low GC content (shown in red and blue, respectively) show similar expression levels in the SMART-Seq v4 and SMART-Seq HT kits (Panel C). Thus, the One-Step RT PCR reaction introduced in the new SMART-seq HT Kit maintains the representation of the low- and high-GC content genes.
High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq HT kits
High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq HT kits. Libraries prepared from 10 pg of Mouse Brain Total RNA shown in Table 1 were further evaluated for the overlap in the number of transcripts identified (FPKM >0.1) between technical replicates within each kit, and found to be very similar (61–63% overlap) (Panel A). Transcripts identified by all three replicates for each kit were then compared against each other, indicating an overlap of 71%. The overlapping transcripts have an average expression level of 37 FPKM, while the transcripts uniquely identified with individual kits are less abundant, averaging between 6–7 FPKM, indicating that the transcripts more likely to not be identified are the ones expressed at a low level.
Schematic of technology in the SMART-Seq HT Kit
Schematic of technology in the SMART-Seq HT Kit. SMART technology is used in a ligation-free workflow to generate full-length cDNA. The reverse transcriptase (RT) adds non-templated nucleotides (indicated by Xs) which hybridize to the SMART-Seq HT Oligonucleotide, providing a new template for the RT. Chemical modifications to block ligation during sequencing library preparation are present on some primers (indicated by black stars). The SMART adapters, added by the oligo (dT) primer (SMART-Seq CDS Primer IIA) and SMART-Seq HT Oligonucleotide and used for amplification during PCR, are indicated in green. The one-step RT-PCR is set up as a single reaction so that all the reagents are mixed together but then used sequentially. SeqAmp polymerase is a hot-start DNA polymerase and is activated only after the reverse transcription/template-switching step is complete.
Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows
Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows. The SMART-Seq v4 method (left) was modified to generate a simplified, high-throughput 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 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 cDNA is used for sequencing library preparation (Nextera XT).
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Gene expression data obtained from FACS-sorted 293T cells
High reproducibility of gene expression data obtained from FACS-sorted 293T cells using the SMART-seq v4 and SMART-Seq HT kits. Libraries generated from twenty-one individual 293T cells (table, top panel) were further analyzed to evaluate the reproducibility of gene expression measurements obtained for each cell with the SMART-Seq v4 kit (SSv4_1 to SSv4_12) and the SMART-Seq HT Kit (HT_1 to HT_9)(lower panel). The hierarchical clustering heat map shows Euclidean distances between all the cells and reports Pearson correlations ranging from 0.74 to 0.97. While the best correlations are observed between cells prepared with one or the other kit, the correlations are still very high between the two kits and the cells are not clustering based on the library preparation method. These data demonstrate that the modified workflow in the SMART-Seq HT Kit does not introduce major bias in measurement of gene expression levels.
Comparison of expression level by gene GC content between the SMART-Seq v4 and SMART-Seq HT kits
Comparison of expression level by gene GC content between the SMART-Seq v4 and SMART-Seq HT kits. The libraries made from 10 pg of Mouse Brain Total RNA shown in the table (Figure 2) were further analyzed for GC content representation (see table, top panel). Genes were binned by GC content, and correlation plots were used to visualize the reproducibility of the expression levels (FPKM) of genes in each bin. The average gene counts are very reproducible for replicate samples analyzed using the SMART-Seq v4 (Panel A) or SMART-Seq HT kits (Panel B). Genes with high or low GC content (shown in red and blue, respectively) show similar expression levels in the SMART-Seq v4 and SMART-Seq HT kits (Panel C). Thus, the One-Step RT PCR reaction introduced in the new SMART-seq HT Kit maintains the representation of the low- and high-GC content genes.
High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq HT kits
High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq HT kits. Libraries prepared from 10 pg of Mouse Brain Total RNA shown in Table 1 were further evaluated for the overlap in the number of transcripts identified (FPKM >0.1) between technical replicates within each kit, and found to be very similar (61–63% overlap) (Panel A). Transcripts identified by all three replicates for each kit were then compared against each other, indicating an overlap of 71%. The overlapping transcripts have an average expression level of 37 FPKM, while the transcripts uniquely identified with individual kits are less abundant, averaging between 6–7 FPKM, indicating that the transcripts more likely to not be identified are the ones expressed at a low level.
Schematic of technology in the SMART-Seq HT Kit
Schematic of technology in the SMART-Seq HT Kit. SMART technology is used in a ligation-free workflow to generate full-length cDNA. The reverse transcriptase (RT) adds non-templated nucleotides (indicated by Xs) which hybridize to the SMART-Seq HT Oligonucleotide, providing a new template for the RT. Chemical modifications to block ligation during sequencing library preparation are present on some primers (indicated by black stars). The SMART adapters, added by the oligo (dT) primer (SMART-Seq CDS Primer IIA) and SMART-Seq HT Oligonucleotide and used for amplification during PCR, are indicated in green. The one-step RT-PCR is set up as a single reaction so that all the reagents are mixed together but then used sequentially. SeqAmp polymerase is a hot-start DNA polymerase and is activated only after the reverse transcription/template-switching step is complete.
Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows
Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows. The SMART-Seq v4 method (left) was modified to generate a simplified, high-throughput 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 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 cDNA is used for sequencing library preparation (Nextera XT).
