SMART-Seq Total RNA Pico Input with and without UMIs (ZapR Mammalian) has been widely used for whole transcriptome profiling, gene expression analysis, coding and long non-coding RNA species detection, post-transcriptional modifications investigation, and gene regulation studies across diverse conditions.

The technology demonstrates excellent performance with compromised RNA (RNA integrity number [RIN] 2–10), such as highly degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples, laser capture microscopy (LCM) samples, and cell-free RNA samples (DV200 >25%).

The workflow adopts SMART® technology for full-length cDNA synthesis and ZapR technology for depletion of ribosomal sequences, generating high-quality RNA-seq libraries from a wide input range of 250 pg–1 µg. 

Sequencing performance of the SMART-Seq Pico Input with UMIs (ZapR Mammalian) and SMART-Seq Pico Input (ZapR Mammalian) kits

For the SMART-Seq Pico Input with UMIs (ZapR Mammalian) kit, libraries were prepared using 10 ng, 100 ng, and 1,000 ng of human brain RNA and sequenced up to 22 M paired-end reads. Five cycles of PCR1 were performed for the 10 and 100 ng input amounts; three cycles of PCR1 were performed for the 1,000 ng input amount. The results were analyzed with Cogent™ NGS Analysis Pipeline (CogentAP).   

For the SMART-Seq Pico Input (ZapR Mammalian) kit, libraries were prepared using 10 ng, 100 ng, and 1,000 ng of human brain RNA and sequenced up to 15 M paired-end reads. Five cycles of PCR1 were performed for the 10 and 100 ng input amounts; three cycles of PCR1 were performed for the 1,000 ng input amount. The results were analyzed with CogentAP.

Figure 1 shows the consistent read distribution for both kits across inputs ranging from 10 ng to 1,000 ng, a high proportion of biologically informative reads, and low unwanted rRNA reads.

Figure 1. SMART-Seq Total RNA Pico Input technology performance for 10 ng, 100 ng, and 1,000 ng input amounts. Panel A. Using the SMART-Seq Total RNA Pico Input with UMIs (ZapR Mammalian) kit, libraries were prepared with human brain RNA and sequenced up to 22 M paired-end reads. Panel B. Using the SMART-Seq Total RNA Pico Input (ZapR Mammalian) kit, libraries were prepared with human brain RNA and sequenced up to 15 M paired-end reads. For both, data were collected and analyzed with the CogentAP to determine performance metrics for the three input amounts. 

Table 1 shows the highly sensitive gene and transcript detection and high strandedness achieved by both kits.   

Table 1. SMART-Seq sensitivity detection performance for 10 ng, 100 ng, and 1,000 ng input amounts using the SMART-Seq Pico Input with UMIs (ZapR Mammalian) and the SMART-Seq Pico Input (ZapR Mammalian) kits. Libraries were prepared with human brain RNA and sequenced up to 22 M paired-end reads for the kit with UMIs and 15 M paired-end reads for the kit without UMIs.

Correlation of gene expression data of the SMART-Seq Pico Input with UMIs (ZapR Mammalian) and SMART-Seq Pico Input (ZapR Mammalian) kits across different input amounts

Transcript expression data across the three different RNA input amounts also showed excellent correlation, indicating the technology’s reliable performance across different input amounts (Figure 2).      

Figure 2. Correlation of transcript expression data between different RNA input amounts. Panel A. Using the SMART-Seq Pico Input with UMIs (ZapR Mammalian) kit, libraries were prepared with 10 ng, 100 ng, and 1,000 ng human brain RNA and sequenced up to 22 M paired-end reads. Each comparison demonstrates the transcript expression correlation across different inputs: 10 ng and 100 ng of RNA (Subpanel A1), 100 ng and 1,000 ng of RNA (Subpanel A2), and 10 ng and 1,000 ng RNA (Subpanel A3). Panel B. Using the SMART-Seq Pico Input (ZapR Mammalian) kit, libraries were prepared with 10 ng, 100 ng, and 1,000 ng human brain RNA and sequenced up to 15 M paired-end reads. Each comparison demonstrates the transcript expression correlation across different inputs: 10 ng and 100 ng of RNA (Subpanel B1), 100 ng and 1,000 ng of RNA (Subpanel B2), and 10 ng and 1,000 ng RNA (Subpanel B3).   

Regardless of RNA input or quality, the SMART-Seq Total RNA Pico Input technologies deliver highly consistent, high-quality data with excellent mapping rates and a high proportion of informative reads, which leads to remarkable sensitivity. With the flexibility to accommodate the widest range of sample types and input amounts, and the accuracy to deliver reliable data every time, these kits serve as a true 'all-in-one' solution—eliminating the need for multiple RNA-seq library prep workflows and the accompanying hassle of choosing from different technologies.

To prepare libraries using >10 ng total RNA, adjust PCR 1 and PCR 2 cycles as indicated in Table 2. 

For all other steps, follow the user manual for the respective kit.