SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing
Brand: Takara Bio.
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SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing
SMART-Seq v4 full-length transcriptome analysis with ultimate sensitivity for ultra-low RNA inputs
The SMART-Seq v4 Kit (SSv4) is the fourth generation of our SMARTer ultra-low solutions and our most sensitive kit for ultra-low inputs. This kit uses oligo(dT) priming to generate high-quality, full-length cDNA directly from multiple intact cells (up to 1,000 cells) or 10 pg–10 ng of total RNA.
The SMART (Switching Mechanism at 5' End of RNA Template) technology powering the cDNA synthesis technology employed by the SMART-Seq v4 kit provides full-length transcript information, enabling analysis of transcript isoforms, gene fusions, point mutations, etc. Additionally, SSv4 takes this technology to the next level by improving upon the Smartseq2 method and incorporating locked nucleic acid (LNA) technology in the SMART-Seq v4 Oligo for more efficient template switching. This allows for the identification of higher numbers of genes relative to other methods. The cDNA kits provide high reproducibility, even gene-body coverage, and an accurate representation of GC-rich transcripts.
The SSv4 PLUS kit includes our patented library preparation kit that incorporates 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 with no intermediate purification steps necessary—minimizing sample mix-up, sample loss, and cross-contamination. The high library yield generated from this kit provides the flexibility to sequence on high-throughput sequencers while providing high reproducibility.
If you are interested in these benefits but are working with intact, high-quality single cells, we recommend our SMART-Seq Single Cell Kit.
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
- 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
- Unparalleled sensitivity—start with as little as 10 pg of total RNA (input range: up to 1,000 cells or 10 pg–10 ng of total RNA)
- High-quality RNA-seq data—identify the highest number of genes for total RNA; obtain full-length transcript information, a low percentage of rRNA reads, and a great representation of GC-rich transcripts
- Increase your 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 system) or confidently save extra library for future runs
Applications
- cDNA synthesis from multiple cells or total RNA for transcriptome sequencing
- cDNA outputs from the SSv4 kit can be used with either the SMART-Seq Library Prep kit (not sold separately; sold as part of the SSv4 PLUS kit) for Illumina sequencing or with the Ion Xpress Plus Fragment Library Kit for Ion Torrent sequencing
- For information on sequencing with the Oxford Nanopore, please contact Scientifix
Reproducibility is high for low-input samples
Reproducibility is high for low-input samples. FPKMs from replicate libraries generated from 10 pg of Mouse Brain Total RNA using the SMART-Seq v4 kit, the SMARTer Ultra Low v3 kit, or the SMART-Seq2 method were compared. For transcripts with FPKM <100, the correlation between replicates was much higher for the SMART-Seq v4 kit (Pearson R = 0.739; Panel B) compared to the SMARTer Ultra Low v3 (Pearson R = 0.376; Panel A) or the SMART-Seq2 method (Pearson R = 0.496; Panel C). For all transcripts (shown in the scatterplots on the right) the correlation between replicates was high for each of the three methods (Pearson R between 0.911–0.972), though the SMART-Seq v4 kit did have the highest correlation. Transcripts represented in only one replicate can be seen along the X- and Y-axes of the scatter plots showing all transcripts.
Gene body coverage is good for all three library preparation methods
Gene body coverage is good for all three library preparation methods. Gene body coverage shown is the average of two replicate libraries prepared from 10 pg Mouse Brain Total RNA using the three different cDNA synthesis methods. The SMARTer Ultra Low v3 kit produced a slight 3′ bias, and the SMART-Seq v4 kit produced a slight 5′ bias; however, the overall coverage was fairly even.
Higher sensitivity and better mappability with the SMART-Seq v4 kit
Higher sensitivity and better mappability with the SMART-Seq v4 kit. Replicate libraries were generated from 10 pg Mouse Brain Total RNA using the SMART-Seq v4 kit, the SMARTer Ultra Low v3 kit, or the SMART-Seq2 method. 18 PCR cycles were used to amplify cDNA libraries with the SMART-Seq2 method and SMARTer Ultra Low v3 kit; however, only 17 PCR cycles were needed for the SMART-Seq v4 libraries.
Sequencing metrics are consistent across RNA input amounts
Sequencing metrics are consistent across RNA input amounts. 10 pg–10 ng of Human Brain Total RNA were used to generate cDNA libraries in duplicate with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing. cDNA libraries were amplified using 17, 14, 10, or 7 PCR cycles for the 10 pg, 100 pg, 1 ng, or 10 ng libraries, respectively.
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Reproducibility is high for low-input samples
Reproducibility is high for low-input samples. FPKMs from replicate libraries generated from 10 pg of Mouse Brain Total RNA using the SMART-Seq v4 kit, the SMARTer Ultra Low v3 kit, or the SMART-Seq2 method were compared. For transcripts with FPKM <100, the correlation between replicates was much higher for the SMART-Seq v4 kit (Pearson R = 0.739; Panel B) compared to the SMARTer Ultra Low v3 (Pearson R = 0.376; Panel A) or the SMART-Seq2 method (Pearson R = 0.496; Panel C). For all transcripts (shown in the scatterplots on the right) the correlation between replicates was high for each of the three methods (Pearson R between 0.911–0.972), though the SMART-Seq v4 kit did have the highest correlation. Transcripts represented in only one replicate can be seen along the X- and Y-axes of the scatter plots showing all transcripts.
Gene body coverage is good for all three library preparation methods
Gene body coverage is good for all three library preparation methods. Gene body coverage shown is the average of two replicate libraries prepared from 10 pg Mouse Brain Total RNA using the three different cDNA synthesis methods. The SMARTer Ultra Low v3 kit produced a slight 3′ bias, and the SMART-Seq v4 kit produced a slight 5′ bias; however, the overall coverage was fairly even.
Higher sensitivity and better mappability with the SMART-Seq v4 kit
Higher sensitivity and better mappability with the SMART-Seq v4 kit. Replicate libraries were generated from 10 pg Mouse Brain Total RNA using the SMART-Seq v4 kit, the SMARTer Ultra Low v3 kit, or the SMART-Seq2 method. 18 PCR cycles were used to amplify cDNA libraries with the SMART-Seq2 method and SMARTer Ultra Low v3 kit; however, only 17 PCR cycles were needed for the SMART-Seq v4 libraries.
Sequencing metrics are consistent across RNA input amounts
Sequencing metrics are consistent across RNA input amounts. 10 pg–10 ng of Human Brain Total RNA were used to generate cDNA libraries in duplicate with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing. cDNA libraries were amplified using 17, 14, 10, or 7 PCR cycles for the 10 pg, 100 pg, 1 ng, or 10 ng libraries, respectively.
