Takara SMART-Seq

SMART-Seq mRNA generates high-quality oligo(dT)-primed, full-length cDNA from ultra-low amounts of total RNA or directly from multiple intact cells (< 1,000 cells). This kit can accommodate an input volume of 10 μl and is regularly tested with 10 pg of total RNA. The kit is available in 24, 96, or 4 x 96 reaction formats.

Reproducibility is high for low-input samples. FPKMs from replicate libraries generated from 10 pg of Mouse Brain Total RNA using SMART-Seq mRNA (an equivalent replacement for SMART-Seq v4), 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 SMART-Seq mRNA 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 mRNA (an equivalent replacement for SMART-Seq v4) produced a slight 5 bias; however, the overall coverage was fairly even.

Higher sensitivity and better mappability with the SMART-Seq mRNA (an equivalent replacement for SMART-Seq v4 kit). Replicate libraries were generated from 10 pg Mouse Brain Total RNA using SMART-Seq mRNA, 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 mRNA 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 mRNA kit (an equivalent replacement for SMART-Seq v4). 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.