The SMARTer Stranded RNA-Seq Kit includes the components needed to generate indexed cDNA libraries suitable for next-generation sequencing (NGS) on any Illumina platform, starting from as little as 100 pg of polyA-purified or ribosomal RNA-depleted RNA. The kit consists of the SMARTer Stranded RNA-Seq Components, SeqAmp DNA Polymerase, and the llumina Indexing Primer Set (PCR primers for the amplification of indexed, paired-end Illumina-compatible sequencing libraries, which enable multiplexing of NGS library analysis).

The SMARTer Stranded RNA-Seq Kit utilizes our patented SMART (Switching Mechanism At 5' end of RNA Template) technology, coupled with PCR amplification, to generate Illumina-compatible libraries without the need for enzymatic clean-up or adapter ligations. The directionality of the template-switching reaction preserves the strand orientation of the RNA, making it possible to obtain strand-specific sequencing data from the synthesized cDNA.

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cDNA libraries produced using the SMARTer Stranded RNA-Seq Kit show comparable yields and purity irrespective of input RNA concentrations

cDNA libraries produced using the SMARTer Stranded RNA-Seq Kit show comparable yields and purity irrespective of input RNA concentrations. Agilent 2100 Bioanalyzer gel-like image using a High Sensitivity DNA chip with cDNA libraries produced from between 100 ng–100 pg human brain polyA⁺ RNA with the number of PCR cycles indicated. cDNA libraries were produced by amplification with SeqAmp DNA Polymerase (included in the SMARTer Stranded RNA-Seq Kit).

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The SMARTer Stranded RNA-Seq Kit provides high reproducibility and sensitivity over a thousand fold range of input RNA levels

The SMARTer Stranded RNA-Seq Kit provides high reproducibility and sensitivity over a thousand fold range of input RNA levels. Scatter plots of expression (FPKM) for cDNA libraries prepared from 100 ng and 100 pg of human brain polyA⁺ RNA show a high correlation, suggesting consistency across input levels. Axes are plotted on a log₁₀ scale. Insets indicate the Pearson coefficient of correlation between replicates (R).

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Data from technical replicates of each library indicate that the SMARTer Stranded RNA-Seq Kit delivers very consistent results across a wide range of input RNA concentrations

Data from technical replicates of each library indicate that the SMARTer Stranded RNA-Seq Kit delivers very consistent results across a wide range of input RNA concentrations. Comparisons of pairs of cDNA library replicas created from 100 ng and 100 pg of input RNA show high reproducibility across a wide range of input RNA. Axes are plotted on a log₁₀ scale. Insets indicate the Pearson coefficient of correlation between replicates (R).

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Even at the lowest levels tested (100 pg polyA+ input RNA), the SMARTer Stranded RNA-Seq Kit was able to maintain high accuracy while detecting ~15,000 genes using strand-specific alignments

Even at the lowest levels tested (100 pg polyA⁺ input RNA), the SMARTer Stranded RNA-Seq Kit was able to maintain high accuracy while detecting ~15,000 genes using strand-specific alignments.

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High reproducibility with the SMARTer Stranded RNA-Seq Kit, confirmed by ERCC analysis

High reproducibility with the SMARTer Stranded RNA-Seq Kit, confirmed by ERCC analysis. Reads mapping to the ERCC data set from the previous experiments were pooled together and the FPKM was plotted against relative transcript abundance. The complete set of 92 ERCC transcripts was identified, with a slope of 0.934 and R² of 0.9725. Axes are plotted on a log₂ scale.

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Indexed cDNA libraries were prepared according to the SMARTer Stranded RNA-Seq Kit protocol using twelve indices, and sequenced on an Illumina HiSeq Platform with 2 x 100 bp paired end reads

Indexed cDNA libraries were prepared according to the SMARTer Stranded RNA-Seq Kit protocol using twelve indices, and sequenced on an Illumina® HiSeq® Platform with 2 x 100 bp paired end reads. Short, overlapping reads originating from different strands of the genomic DNA were distinguished from each other, enabling quantitative expression analysis and accurate genome annotation. 99% of RNA-Seq reads mapped to the correct strand.

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Distinguishing overlapping and antisense transcripts with the SMARTer Stranded RNA-Seq Kit

Distinguishing overlapping and antisense transcripts with the SMARTer Stranded RNA-Seq Kit. Panel A. RNA-seq reads from the Human Brain Poly A+ RNA cDNA library were mapped against the human genome. The SMARTer Stranded method allowed assignment of sequencing reads to the correct gene in the case of overlapping PHC1 and M6PR transcripts. Panel B. Strand-specific coverage of the CDR1 locus. Nearly all reads are antisense to the annotated transcript, a finding independently reported elsewhere (Hansen, T. B. et al. (2011) EMBO J. 30(21):4414–4422). Panel C. Comparison of CDR1 gene counts obtained using either a strand-agnostic or strand-aware method.

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RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis

RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis. RNA-seq libraries were generated from Human Brain Total RNA (Clontech) or Breast Cancer FFPE RNA (Cureline, extracted using a NucleoSpin totalRNA FFPE kit) and treated with the indicated rRNA removal method. Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-Seq Metrics. Libraries generated from RiboGone-treated RNA had comparably low rRNA reads to oligo(dT)-enriched RNA (Clontech), while retaining more noncoding reads.

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634838: SMARTer Stranded RNA-Seq Kit