SMARTer smRNA-Seq Kit for Illumina

Demonstrating the accuracy of the SMARTer approach for small RNA-seq

Demonstrating the accuracy of the SMARTer approach for small RNA-seq. Sequencing libraries were generated from an equimolar pool of 963 synthetic miRNAs (miRXplore Universal Reference) using the SMARTer smRNA-Seq Kit for Illumina (1 ng input; purple), or a small RNA-seq kit from a different vendor (Competitor N) employing an adapter ligation method (100 ng input; blue). Following sequencing, mapping, and counting of reads, miRNA expression levels (Y axis, log scale) were normalized, resulting in an expected expression level equal to 1 for each miRNA, and a 2-fold cutoff was assigned both above and below the expected expression level (indicated by two horizontal lines). For visualization purposes, miRNAs are ranked along the X axis in order of expression level.

Reproducibility of SMARTer small RNA-seq data

Reproducibility of SMARTer small RNA-seq data. Sequencing libraries were generated in parallel from the indicated input amounts of human brain total RNA using the SMARTer smRNA-Seq Kit for Illumina, and size selected using the BluePippin system. Following sequencing, data processing, and mapping, expression levels of miRNAs identified for each library were quantified and plotted on correlation diagrams. Panel A. Correlation of miRNA expression levels for experimental replicates involving 1 ng inputs. Panel B. Correlation of miRNA expression levels for 2 µg vs. 1 ng inputs.

Schematic of technology used by the SMARTer smRNA-Seq Kit for Illumina

Schematic of technology used by the SMARTer smRNA-Seq Kit for Illumina. SMART technology is used in a ligation-free workflow to generate sequencing libraries for Illumina platforms. Input RNA is first polyadenylated in order to provide a priming sequence for an oligo(dT) primer. cDNA synthesis is primed by the 3’ smRNA dT Primer, which incorporates an adapter sequence (green) at the 5’ end of each first-strand cDNA molecule. When the MMLV-derived PrimeScript Reverse Transcriptase (RT) reaches the 5’ end of each RNA template, it adds non-templated nucleotides which are bound by the SMART smRNA Oligo—enhanced with locked nucleic acid (LNA) technology for greater sensitivity. In the template-switching step, PrimeScript RT uses the SMART smRNA Oligo as a template for the addition of a second adapter sequence (purple) to the 3’end of each first-strand cDNA molecule. In the next step, full-length Illumina adapters (including indexes for sample multiplexing) are added during PCR amplification. The Forward PCR Primer binds to the sequence added by the SMART smRNA Oligo, while the Reverse PCR Primer binds to the sequence added by the 3’ smRNA dT Primer. Resulting library cDNA molecules include adapters required for clustering on an Illumina flow cell (P5 shown in light blue, P7 shown in red), Illumina TruSeq® HT indexes (Index 2 [i5] shown in orange, Index 1 [i7] shown in yellow), and regions bound by the Read Primer 1 or Read Primer 2 sequencing primers (shown in purple and green, respectively). Note that adapters included in the final library add 153 bp to the size of RNA-derived insert sequences.