Ovation^® SoLo RNA-Seq Library Preparation Kit

What materials are provided with the Ovation SoLo RNA-Seq System?

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 NuGEN will provide AnyDeplete primers with the Core kit.

Can this system be used with other library preparation workflows?

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.

What equipment is required or will be useful?

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 Tapestation may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B of the User Guide.

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

• Norgen Biotek Total RNA Purification Kit
• Zymo Research Quick-RNA™ Kits
• Arcturus PicoPure^® RNA Isolation Kit
• Ambion PureLink^® RNA Mini Kit
• Qiagen RNeasy Kits

For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:

• Norgen Biotek FFPE RNA Purification Kit
• Zymo Research Quick-RNA™ FFPE Kit
• Arcturus^® Paradise^® PLUS FFPE RNA Isolation Kit
• PureLink™ FFPE RNA Isolation Kit
• Qiagen RNeasy FFPE Kit

Organic methods such as TRIzol^® Reagent should be subsequently followed with a column-based clean-up method.

Can I use TRIzol^® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol^® or similar methods as any carry over 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.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Technical Support for more information.

Can I use Ovation SoLo RNA-Seq System with RNA from any organism?

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 NuGEN Technical Support.

Do I need to use high-quality total RNA?

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.

Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Ovation SoLo RNA-Seq System?

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.

Can I sort my cells into PBS or another buffer prior to beginning the cell lysis protocol?

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 may inhibit reactions in the kit and yield variable sequencing results. Contact NuGEN Technical Support for additional information.

What cell types are compatible with the lysis buffer provided with the kit?

The lysis buffer has been tested with cell inputs between 1–500 cells and should be compatible with most dissociated cells. Some direct cell inputs may need to be optimized depending on cell type. Decreased performance may be observed with cell types containing excessive ipids, 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.

Can contaminating genomic DNA interfere with Ovation SoLo RNA-Seq System performance?

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.

Does this system contain a SPIA^®-based amplification?

No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.

Does cDNA generated with the Ovation SoLo RNA-Seq System require fragmentation?

No, fragmentation of cDNA occurs during the cDNA Processing steps in the protocol.

Can I combine the barcoded libraries prior to the PCR amplification step?

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 quantitated independently before being pooled for use on the sequencer.

Where can I safely stop in the protocol?

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.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

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.

What magnetic separation devices do you recommend for the SPRI bead purifications?

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:

• Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
• Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
• Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

• Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
• Ensure that the beads are fully resuspended in solution before adding to the sample.
• Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
• Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

How do I measure my final library yield? Can I use an Agilent Bioanalyzer to evaluate the product?

Please refer to section V.Q of the User Guide for guidelines on quantitative and qualitative assessment. We recommend using a qPCR based-method in combination with the Agilent Bioanalyzer or Tapestation for the most accurate quantification.

Are irregular Bioanalyzer traces normal for libraries generated with the Ovation SoLo RNA-Seq System?

Yes, irregular Bioanalyzer traces are normal for libraries generated with the Ovation SoLo RNA-Seq System.

What is the average size of the library generated by 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.

How many bases do Ovation SoLo RNA-Seq System adaptors add to the library?

The adaptors add 132 bp to the Ovation SoLo RNA-Seq System library.

Is the Ovation SoLo RNA-Seq System compatible with all Illumina sequencing platforms?

Illumina may not support the use of a custom sequencing primer or low diversity libraries on all platforms. Please follow the custom primer and low diversity library recommendations for your specific sequencer.

How much material should I load into the sequencer?

Please follow manufacturer’s recommendations for library QC, quantitation, balancing and loading of the amplified library on the sequencer.

Can the Ovation SoLo RNA-Seq System be used with paired-end sequencing?

Yes, it can be used for both single end 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.

What kind of sequencing primers can I use with your library?

Ovation SoLo RNA-Seq System generated libraries are designed to use standard Illumina sequencing primers for both single end and paired-end sequencing applications when multiplexed with standard Illumina libraries (see Section III.D., Sequencing Recommendations and Guidelines for more information). For dedicated Ovation SoLo RNA-Seq Sequencing runs, the Ovation SoLo Custom R1 primer must be used in place of the Illumina forward read primer. It is provided at a concentration of 100 μM. Due to the use of the Ovation SoLo Custom R1 primer, a PhiX control should not be included in the sequencing run.

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

Each eight-base 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.

Do you recommend trimming sequences before downstream analysis?

Yes. We recommend trimming the 5’ end of the sequence as recommended in Section III.F. Data Analysis 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.

Can I use standard alignment algorithms to analyze strand-specific sequencing data?

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.

What percentage of rRNA reads can I expect in my data?

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).

Custom depletion designs can be tailored to any transcript, any organism. Please see our page on custom AnyDeplete designs for more information.

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