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Streamlined solution for mRNA-Seq library preparation for sequencing on Illumina sequencers. This kit features a broad input range, UDI, and simple library quantification with NuQuant.
The Universal Plus mRNA-Seq with NuQuant is now available with up to 384 UDI (Unique Dual Index) adaptors for maximum multiplexing flexibility to optimize your sequencing runs.
Standard whole transcriptome RNA-Seq captures both informative and non-informative data. mRNA-Seq is a more targeted approach for studying the transcriptional coding regions and capturing highly informative gene expression data that can be used in multiple applications.
mRNA-Seq is used to examine transcriptional expression in a wide variety of research areas, including disease conditions. mRNA-Seq is a selective RNA-Seq procedure that enriches for all poly(A) transcripts within the transcriptome. Targeting mRNA maximizes sequencing coverage since resources are dedicated to the sequencing of coding regions. This enables the detection of rare variants and low expressed mRNA transcripts.
Universal Plus mRNA-Seq kit with NuQuant® library quantification is a novel and streamlined library preparation solution for mRNA sequencing on Illumina sequencers. This kit is designed to provide poly(A) selection efficiency and uniform coverage for a broad range of RNA inputs. This RNA-Seq library preparation kit is integrated with NuQuant, a proprietary library quantification method for efficient and accurate quantitation of Next-Generation Sequencing (NGS) libraries. The NuQuant method eliminates the need for time-consuming or inaccurate library quantification methods like qPCR, fluorometry (e.g. Qubit®) or microfluidic electrophoresis (e.g. Bioanalyzer®) allowing RNA-Seq libraries to be constructed and quantified in a single day.
NuQuant app for Qubit® can be downloaded here on Github.
NuQuant Library Quantification provides a simple library quantification that saves time and money. During library preparation, each library molecule has an equivalent number of labels incorporated, regardless of the size of the library fragment, resulting in direct measurement of the molar concentration using standard fluorometers. NGS library quantification with NuQuant is accurate, easy to use and is integrated with the Universal mRNA-Seq with NuQuant library preparation kit.
NuQuant has the lowest variability during library quantitation by multiple users. A set of 8 libraries was distributed to 6 different users at multiple institutions. Users were asked to use NuQuant and their own preferred method of library quantitation (number denoted by n). For each library, and each quantification method, the percent coefficient of variation (%CV) of molar concentration was calculated. NuQuant produced the lowest variation, followed by qPCR. Bioanalyzer gave the highest variation.
Strong correlation between NuQuant molarity and sequencing read numbers. Two users prepared 8 libraries each from 10 ng of Covaris fragmented genomic DNA and amplified the libraries with either 10 or 13 cycles to produce both lower and higher library concentrations. The 16 purified libraries were quantified by NuQuant and equal volumes of all libraries were pooled and sequenced on one lane of an Illumina sequencer. The number of reads from each library correspond to the library molarity determined by NuQuant demonstrating the accuracy of quantification.
NuQuant simplifies library quantification. Using any standard fluorometer, NuQuant generates library molarity values that can be used for pooling NGS libraries before sequencing.
NuQuant saves time and money by eliminating the need for qPCR and bioanalyzer steps and reagents.
Universal Plus mRNA-Seq library preparation kit with NuQuant was used to prepare Illumina libraries starting with 10 ng, 100 ng and 1 μg of K562 total RNA. Consistent library metrics regardless of input was observed. This allows for RNA sequencing of low abundant samples that were previously inaccessible.
Universal Plus mRNA-Seq library preparation kit with NuQuant provides consistent results regardless of input. Similar detection of RefSeq genes even at lower inputs, demonstrating robust performance and high quality data regardless of input.
Universal Plus mRNA-Seq library prep kit with NuQuant provides even coverage across the entire transcript enabling detection of splice variants and isoforms during data analysis.
Universal Plus mRNA-Seq library prep kit was used to construct Illumina libraries from 500 ng of total RNA from fetal cord blood. In the absence of AnyDeplete-mediated globin transcript depletion, globin represented ~77% of the sequencing reads.
Addition of adult globin depletion reduced the percent of globin down to ~43%, increasing the number of informative reads. The AnyDeplete probe mix was customized to target fetal globin which reduced the goblin to ~10%. Eliminating unwanted globin reads resulted in the detection of over a 1000 more RefSeq genes providing more complete data.
Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq
10 ng - 1 μg
Up to 384-plex with Unique Dual Index adaptors
The Universal Plus mRNA-Seq core kit provides all necessary buffers, primers and enzymes for poly(A) selection and library construction. SPRI purifcation beads and EvaGreen are not included. Reagents for AnyDeplete are available separately.
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.
Universal Plus mRNA-Seq is an end-to-end solution designed to generate libraries for Illumina sequencing starting from total RNA and has not been tested with alternative library preparation systems.
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.
Universal Plus mRNA-Seq has been designed for use with total RNA inputs containing polyadenylated transcripts. Universal Plus mRNA-Seq is compatible with polyadenylated RNA from any organism.
Yes, high-quality RNA is required for efficient poly(A) selection.
Contaminating genomic DNA can be incorporated into libraries and inclusion of a DNase treatment during RNA isolation is recommended.
No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.
No. Chemical fragmentation is incorporated into the workflow after poly(A) RNA selection.
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 second strand synthesis, end repair, strand selection 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 the User Guide section 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.
Single index adaptors add 122 bp to the library. Dual index adaptors add 144 bp to the library.
Universal Plus mRNA-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.
Universal Plus mRNA-Seq libraries are designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.
Yes. The libraries produced using this kit 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. Contact Tecan NGS Technical Support for additional information.
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. 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 Universal Plus mRNA-Seq, the forward read corresponds to the sense strand.
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For research use only. Not for use in diagnostic procedures.