Takara SMART-Seq® mRNA HT

SMART-Seq mRNA HT is an automation-friendly 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). cDNA libraries generated with this kit have been tested for compatibility with Illumina sequencing platforms.This kit supports up to 24 reactions.

Comparison of the SMART-Seq mRNA HT (equivalent replacement for the SMART-Seq HT Kit) and SMART-Seq mRNA (equivalent replacement for SMART-Seq v4) workflows.The SMART-Seq v4 method (left) was modified to generate a simplified, high-throughput workflow (SMART-Seq HT Kit, 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 additional 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). Please note thatSMART-Seq mRNA HT is an equivalent replacement for the SMART-Seq HT Kit and SMART-seq mRNA is an equivalent replacement for SMART-Seq v4, and use the same workflows, respectively.

Schematic of technology in SMART-Seq mRNA HT (equivalent replacement for 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. Please note thatSMART-Seq mRNA HT is an equivalent replacement for the SMART-Seq HT Kit, and uses the same workflow.

High overlap of transcripts identified with SMART-seq mRNA HT (equivalent replacment for the SMART-Seq HT Kit) and SMART-Seq mRNA (equivalent replacment for SMART-Seq v4).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.Data is shown for the the SMART-Seq HT Kit and SMART-Seq v4.SMART-Seq mRNA HT is an equivalent replacement for the SMART-Seq HT Kit and SMART-Seq mRNA is an equivalent replacment for SMART-Seq v4.

Comparison of expression level by gene GC content between SMART-Seq mRNA HT (equivalent to the SMART-Seq HT Kit) and SMART-Seq mRNA (equivalent to SMART-Seq v4).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.Data is shown for the SMART-Seq HT Kit and SMART-Seq v4.SMART-Seq mRNA HT is an equivalent replacement for the SMART-Seq HT Kit and SMART-Seq mRNA is an equivalent replacement for SMART-Seq v4.

High reproducibility of gene expression data obtained from FACS-sorted 293T cells using SMART-Seq mRNA (equivalent replacement for SMART-Seq v4) and SMART-Seq mRNA HT (equivalent replacement for the SMART-Seq HT Kit).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.Data is shown for the SMART-Seq v4 and SMART-Seq HT Kit.SMART-Seq mRNA HT is an equivalent replacement for the SMART-Seq HT Kit and SMART-Seq mRNA is an equivalent replacment for SMART-Seq v4.