Universal Prokaryotic RNA-Seq Library Preparation Kit

What materials are provided with the Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems?

The Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems provide all necessary buffers, primers, enzymes and SPRI purification beads for generation of Illumina-compatible sequencing libraries. The kit also provides nuclease-free water for purification elution steps.

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

No. The cDNA is generated with selective primers, but no SPIA-based amplification is used.

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.

Can this system be used with other library preparation workflows?

The Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems are an end-to-end solution designed to generate libraries for Illumina sequencing starting from total RNA and have not been tested with alternative library preparation systems.

Can I use the Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems with RNA from any organism?

This system has been designed specifically for prokaryotes. Performance with other organisms may vary.

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

• Norgen Biotek Total RNA, Soil RNA, or Stool RNA Purification Kits
• Zymo Research QuickRNA™ Kit, ZR Fungal/Bacterial RNA MicroPrep™, or ZR Soil/Fecal RNA MicroPrep™
• Arcturus PicoPure^® RNA Isolation Kit
• Ambion PureLink^® RNA Mini Kit
• Qiagen RNeasy Kits

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.

Will the use of RNA purification columns impact my data?

We have observed changes in alignment metrics and expression profiles with the use of purification columns such as the QIAGEN RNeasy column. We recommend consulting the manufacturer to ensure the RNAs of interest are retained after purification.

Do I need to use high-quality total RNA?

The Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems are designed to work with purified total RNA. When using purified total RNA, 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 Complete Prokaryotic RNA-Seq DR Multiplex Systems?

The system is designed to use total RNA as input. This system utilizes NuGEN’s customizable AnyDeplete technology to deplete targeted transcripts. rRNA depletion or poly(A) enrichment is not necessary.

How much total RNA do I need for amplification?

The selective priming process is designed to deplete rRNA from 100–500 ng total RNA input.

Can contaminating genomic DNA interfere with the Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems performance?

Yes, contaminating genomic DNA may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification. For an explanation of DNase requirements see section III.A.4. For DNase treatment of RNA samples, refer to Appendix A for guidelines.

How does your protocol enable strand retention?

The Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems utilize targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.

Does NuGEN provide reagents for performing the fragmentation step of the protocol?

We recommend using the Covaris instrument for cDNA fragmentation, as suggested in the “Materials” section of the product User Guide. NuGEN does not provide the reagents used in the fragmentation steps. Please see section V.C. in the User Guide for recommendations for fragmentation with the Covaris instrument.

I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of these systems. Other means of fragmentation, such as sonication or enzymatic fragmentation, may be suitable as long as the method generates a tight size distribution of cDNA fragments with a median size of 200 bp.

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 can be placed in short-term storage at –20°C after B. Second Strand Synthesis, after C. cDNA Fragmentation or after any of the bead purification steps.

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.O 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.

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

The adaptors add 122 bp to the library.

What sequencers are compatible with your libraries?

Ovation Complete Prokaryotic RNA-Seq Systems libraries are compatible with Illumina sequencing platforms.

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.

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

For experiments using the Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems with dedicated read barcodes, please follow the Illumina recommendations on parsing barcodes. The NuGEN dedicated read barcodes are six-base unique barcode tags. The sequences of these NuGEN barcodes must be input prior to parsing.

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

The Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems are designed for use with the standard Illumina sequencing primers for both single end and paired-end sequencing applications.

Can the Ovation Complete Prokaryotic RNA-Seq DR Multiplex Systems be used with paired-end sequencing?

Yes, they 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 libraries with fragments of an average size of 150 bases, corresponding to the expected distance between the 5´-most and 3´-most coordinates of paired-end reads.

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 Complete Prokaryotic RNA-Seq System, the forward read corresponds to the sense strand.

Will the presence of extrachromosomal material in total RNA impact my data?

It is possible to see a higher proportion of unmapped reads in the context of some bacterial strains with extrachromosomal content, such as plasmids.