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Streamlined Prokaryotic RNA-seq library prep kit for strand-specific RNA-Seq library construction using between 100 ng to 500 ng of total RNA obtained from pure cultures or mixed populations of bacteria.
The Universal Prokaryotic RNA-Seq, Prokaryotic AnyDeplete® library preparation kit is an easy-to-use add and incubate system ideal for microbiological RNA sequencing studies. The Universal Prokaryotic RNA-Seq kit provides a complete solution for strand-specific RNA-Seq library construction using between 100 ng to 500 ng of total RNA obtained from pure cultures or mixed populations of bacteria. The integrated AnyDeplete technology enables rRNA depletion from a broad range of prokaryotes optimizing informative reads and reducing sequencing costs.
Sample types include purified RNA from bacterial cell lines, environmental samples, and microbiome studies.
The Universal Prokaryotic RNA-Seq kit is designed to provide consistent data and performance across diverse bacterial communities or cell cultures.
The composition of NGS libraries is determined by both the efficiency of depletion and the amount of rRNA initially present in the sample. For example, removing 90% of the rRNA from a sample containing an initial load of 98% rRNA and 2% other transcripts results in an NGS library with 17% non-rRNA (9.8% rRNA and 2% mRNA). When the same depletion rate is applied to a sample with 95% rRNA, the non-rRNA component will increase to 34%.
Data was generated with the Universal Prokaryotic RNA-Seq Library Systems or a random primer method, using 100 ng of total RNA input from the indicated sources. The resulting libraries were sequenced on the Illumina Genome Analyzer IIx using 4 libraries per lane (4-plex) with 40 bp single-read sequencing.
AnyDeplete is a flexible solution for the removal of unwanted transcripts from your NGS libraries. Use the standard Prokaryotic AnyDeplete probe mix to remove rRNA from a broad range of bacterial species or customize the AnyDeplete probe mix for your model organism or mix probes together to target multiple species for host-pathogen studies.
Custom targeted depletion of P. gingivalis rRNA reduces the percent of rRNA reads from 90% to 10% providing more useful information with the same number of sequenced reads.
AnyDeplete is able to efficiently eliminate rRNA reads from the library significantly reducing the number of uninformative reads, providing more useful data and lower sequencing costs
AnyDeplete efficiently reduces the amount of unwanted rRNA reads from standard mouse and Drosophila samples and can be customized to target unwanted reads from any organism such as P. gingivalis
Available custom AnyDeplete probes for Universal RNA-Seq with NuQuant library prep kit
The NuQuant workflow for quantification of library molar concentration. Library molarity can be directly measured in less than 6 minutes using a Qubit with the appropriate NuQuant App or a standard fluorescent plate reader
A set of 8 libraries were distributed to 6 different users at multiple institutions. For each library and quantification method, the percent coefficient of variation (% CV) of molar concentration was calculated. NuQuant demonstrated the lowest variation across all users
Sixteen libraries with varying insert sizes and concentrations were quantified with NuQuant. The libraries were then pooled by volume and sequenced. The graph shows the strong correlation between library molarity measured by NuQuant and the number of reads per sample demonstrating the accuracy of NuQuant
Illumina NovaSeq, HiSeq, MiSeq, NextSeq, MiniSeq
Total RNA isolated from bacterial sources
100 ng – 500 ng
The Universal Prokaryotic RNA-Seq kit provides 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.
No. The cDNA is generated with selective primers, but no SPIA-based amplification is used.
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.
The Universal Prokaryotic RNA-Seq kit is 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.
This system has been designed specifically for prokaryotes. Performance with other organisms may vary.
We recommend a column-based method, including:
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
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.
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.
The Universal Prokaryotic RNA-Seq kits 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.
The system is designed to use total RNA as input. This system utilizes Tecan's customizable AnyDeplete technology to deplete targeted transcripts. rRNA depletion or poly(A) enrichment is not necessary.
The selective priming process is designed to deplete rRNA from 100–500 ng total RNA input.
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. For DNase treatment of RNA samples, refer to Appendix A for guidelines.
The Universal Prokaryotic RNA-Seq kit utilizes targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.
Tecan does not provide the reagents used in the fragmentation steps. We recommend using the Covaris instrument for cDNA fragmentation, as suggested in the “Materials” section of the product User Guide.
Please see section V.C. in the User Guide for recommendations for fragmentation with the Covaris instrument.
We have evaluated only Covaris fragmented DNA during the development of these systems. Other sonication instruments or enzymatic fragmentation may be suitable as long as the method generates a tight size distribution of cDNA fragments with a median size of 200–300 bp.
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 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.
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.N 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 Fragment Analyzer for the most accurate quantification.
The adaptors add 124 bp to the library.
Universal Prokaryotic RNA-Seq libraries are compatible with Illumina sequencing platforms.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
The Universal Prokaryotic RNA-Seq kits are designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.
Yes, they 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.
For experiments using the Universal Prokaryotic RNA-Seq, please follow the Illumina recommendations on parsing barcodes. The sequences of the Tecan barcodes must be input prior to parsing.
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 Universal Prokaryotic RNA-Seq, the forward read corresponds to the sense strand.
It is possible to see a higher proportion of unmapped reads in the context of some bacterial strains with extrachromosomal content, such as plasmids.
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