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The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing is the fourth generation of SMARTer® Ultra Low products and generates high-quality cDNA from ultra-low amounts of total RNA, or directly from 1–1,000 intact cells. This kit can accommodate an input volume of 10 µl and is regularly tested with 10 pg of total RNA. It also performs robust cDNA synthesis from a single cell (equivalent to 10 pg of total RNA), generating data with the highest sensitivity compared to previous generations.
Brand: Takara
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The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing is the fourth generation of SMARTer Ultra Low products and generates high-quality cDNA from ultra-low amounts of total RNA, or directly from 1–1,000 intact cells. This kit can accommodate an input volume of 10 μl and is regularly tested with 10 pg of total RNA. It also performs robust cDNA synthesis from a single cell (equivalent to 10 pg of total RNA), generating data with the highest sensitivity compared to previous generations. This kit incorporates locked nucleic acid (LNA) technology and improves on the SMART-Seq2 method. cDNA libraries generated by this kit have been tested for compatibility with Ion Torrent and Illumina sequencing platforms. SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts. Most importantly, SMART technology enriches for full-length transcripts and maintains the true representation of mRNA transcripts from the original sample. These factors are critical for transcriptome sequencing and gene expression analysis.
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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 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. 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. 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.
