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Complete end-to-end sample to library prep solution for stranded, whole transcriptome RNA-Seq analysis of ultra-low inputs from exosomes, FFPE samples and cell-free RNA.
The Ovation® SoLo RNA-Seq System for ultra-low input RNA sequencing is a flexible end-to-end library preparation solution producing strand-specific, rRNA depleted libraries from 10 pg to 10 ng of total RNA.
Utilizing the highly flexible and customizable AnyDeplete technology, this ultra-low input RNA-Seq kit offers depletion of rRNA and other high-abundant transcripts to increase the dynamic range, reduce sequencing costs, and simplify data analysis.
Sample types include purified RNA or lysates from cell lines including fresh or FFPE tissue, liquid biopsy, cell-free RNA and other ultra-low input RNA samples.
Libraries generated from 5 ng of cell-free plasma RNA with a DV200 score of 10-20 %. Analysis of the whole transcriptome provides complete information from coding, non-coding and regulatory transcripts.
Ovation SoLo RNA Seq provides highly correlated data between fresh frozen and FFPE adenocarcinoma samples.
High correlation between technical replicates from inputs as low as 10 pg of total RNA to 1ng, giving you confidence in your data.
Illumina HiSeq, MiSeq, NextSeq, MiniSeq
Total RNA, FFPE tissue, Cell-free RNA
10 pg – 10 ng
The Ovation SoLo RNA-Seq System provides all necessary buffers, primers and enzymes necessary for library construction from cell lysates or isolated RNA. The kit also provides DNA Resuspension Buffer for purification elution steps. This kit does not provide EvaGreen used for Library Amplification qPCR nor the Agencourt RNAClean XP or AMPure XP Beads required for the purification steps. AnyDeplete probes used for targeted transcript depletion are available separately or in a bundle with the Core Kit. For custom designs Tecan will provide AnyDeplete probes with the Core kit.
There is no need to combine the workflow with other library preparation workflows. The Ovation SoLo RNA-Seq System is an end-to-end solution designed to generate Illumina-compatible libraries starting from cells or total RNA.
Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a magnetic plate for 0.2 mL tubes, strips, or plates and a spectrophotometer or fluorometer. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B.
We recommend a column-based method, including:
For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method. Isolation methods that capture RNAs shorter than 200 nt may result in increased 5S and 5.8S rRNA. Do not use carrier RNA during isolation.
We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.
We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.
The Ovation SoLo RNA-Seq System should be suitable for total RNA input from any organism. For information on organism specific rRNA or targeted transcript depletion contact Tecan NGS Technical Support.
When using with the Ovation SoLo RNA-Seq System, samples should be of high molecular weight with little or no evidence of degradation. While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of somewhat degraded samples. With such samples, users may experience lower yields and may encounter affected sequencing metrics.
No. The system is designed to use total RNA as input. rRNA depletion or poly(A) enrichment are not necessary. rRNA, as well as other transcript types, can be targeted for depletion using the AnyDeplete technology embedded in the workflow.
For best results we recommend sorting cells directly into the Lysis Buffer Master Mix prepared with the kit, or resuspending cell pellets in the Lysis Buffer Master Mix. Other buffers (e.g. sheath fluid) may inhibit reactions in the kit and yield variable sequencing results. It is recommended for other buffers not to exceed 1 ul. Contact Tecan NGS Technical Support for additional information.
The lysis buffer has been tested with mammalian cell inputs up to 500 cells and should be compatible with most dissociated cell types. Some direct cell inputs may need to be optimized depending on cell type. Decreased performance may be observed with cell types containing excessive lipids, minerals, collagen, etc. or a cell wall, or with very large cells. Non-dissociated cells, tissue, or LCM samples may require additional optimization of the workflow.
Yes, contaminating genomic DNA may be incorporated into libraries. For this reason DNase treatment is incorporated into both the Cell Lysis and Total RNA Input workflows.
No, fragmentation of cDNA occurs during the cDNA Processing steps in the protocol.
No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.
This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantified independently before being pooled for use on the sequencer.
Samples may be stored at –80 °C after Cell Lysis. Samples may also be placed in short-term storage at –20 °C after First Strand cDNA Synthesis, End Repair, Adaptor Ligation, Library Amplification, or after any purification step.
RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.
Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:
Please refer to section V.Q of the User Guide for guidelines on quantitative and qualitative assessment. A qPCR based-method is required in combination with the Agilent Bioanalyzer or Fragment Analyzer.
Yes, irregular Bioanalyzer traces are normal for libraries generated with the Ovation SoLo RNA-Seq System.
Ovation SoLo RNA-Seq libraries are 300–350 bp in length for high-quality human samples and may be shorter for FFPE and other degraded samples. Libraries generated from model organism samples may also be longer or shorter than human libraries. Take care not to overestimate library size for loading on the sequencer.
The adaptors add 132 bp to the Ovation SoLo RNA-Seq System library.
Special sequencing parameters may be required depending on the Illumina sequencing platform used. Contact Tecan NGS Technical Support for recommendations.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
Yes, it can be used for both single- and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The workflow generates library fragments with an average size of 330 bases.
Ovation SoLo RNA-Seq libraries are designed to use standard Illumina sequencing primers for both single- and paired-end sequencing when multiplexed with standard Illumina libraries (see User Guide section Sequencing Recommendations and Guidelines for more information). For dedicated Ovation SoLo RNA-Seq sequencing runs, contact Tecan NGS Technical Support for recommendations.
Each barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.
Yes. We recommend trimming the 5’ end of the sequence as recommended in the Data Analysis section of the User Guide. This portion of the read corresponds to the overhang of the forward adaptor and may not accurately reflect the biological sequence.
Yes. Strand-specific reads can be processed and mapped to reference sequences using the same methods used for other RNA-Seq libraries. Note that in libraries generated by the Ovation SoLo RNA-Seq System, the forward read corresponds to the sense strand.
The number of rRNA reads present in the sequencing results is dependent on the abundance of rRNA transcripts in the starting material. For a sample containing 10% mRNA and 90% rRNA, a 90% depletion of rRNA transcripts results in a sample containing 53% mRNA and 47% rRNA (i.e. 10% and 9% of the original pool of RNA, respectively).
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For research use only. Not for use in diagnostic procedures.