SMARTer Target RNA Capture for Illumina is a complete system is designed to generate cDNA from enriched total RNA. Targeted RNA-seq can overcome several challenges inherent in whole transcriptome amplification (WTA) RNA-seq experiments, improving sequence coverage and sensitivity of detection of transcripts of interest that may be present in low amounts. Targeted RNA-seq approaches are also advantageous in saving costs and simplifying analysis. Enrichment of transcripts of interest prior to cDNA synthesis enables the capture of information about transcripts that could otherwise be missed or require a much greater number of sequencing reads to be detected, including chimeric gene fusions, transcript isoforms, and splice variants. By improving sensitivity, the power of analysis is also improved, as seen in a reduction in the signal-to-noise ratio.
In the SMARTer Target RNA Capture for Illumina workflow, total RNA is hybridized with user-designed biotinylated DNA probes in less than four hours, and then purified prior to capture on Capture Beads—magnetic particles coated with streptavidin. The Capture Beads have been selected because they exhibit especially low adsorption of protein and nucleic acids, and do not interfere with downstream reactions such as PCR or reverse transcription, making it possible to perform cDNA synthesis on the targeted samples while they remain attached to the beads. Capture Beads are provided as part of the SMARTer Target RNA Capture for Illumina kit, and are also sold separately (Cat. # 635039).
Taking advantage of the sensitivity of the SMART-Seq v4 Ultra Low Input Kit for Sequencing, this kit generates high-quality, full-length cDNA from the enriched transcripts. This kit is designed to work with inputs as low as 10 ng of total RNA as starting material. We have been able to generate successful data with as little as 100 pg of total RNA input; please see the tech note to view the data. This method utilizes oligo(dT) priming and is designed to work with high-quality samples. It does not require additional rRNA removal methods or kits, and generates cDNA libraries compatible with Illumina sequencing platforms.