Dear Customers,

SENSE mRNA-Seq Library Prep Kits (Cat. No. 001.24 and 001.96) are being discontinued and available until stocks last. We recommend instead to use our CORALL mRNA-Seq Library Prep Kit.


SENSE mRNA-Seq Library Prep Kit

SENSE is a complete strand-specific mRNA-Seq library prep kit for accurate gene expression profiling, transcriptome sequencing, discovery and quantification of antisense transcripts and overlapping genes.

Superior Strand-Specificity

The strand-displacement stop/ligation technology used in SENSE generates fewer antisense artifacts which can be produced by template-switching in protocols which utilize RNA or cDNA fragmentation. This results in exceptional (>99.9 %) strand-specificity and reduced experimental noise, enabling the detection and quantification of antisense transcripts with high confidence.

Rapid Turnaround

NGS-ready libraries can be produced from total RNA samples in under 5 hours with less than 50 % hands-on time, allowing RNA extraction, library preparation and quality control to be performed in one day.

All-in-One Solution

No additional kits or reagents for poly(A) RNA selection, library amplification, size selection or purification, or barcodes are required.

Low Amount of Input Total RNA

The typical amount of input total RNA is 1 ng – 2 µg.

Efficient rRNA Elimination

SENSE mRNA-Seq Library Prep includes the Poly(A) RNA Selection Kit (Cat. No. 039) which virtually eliminates cytoplasmic ribosomal RNA (<0.001 % of total reads from Universal Human Reference RNA) and removes the need for additional selection or depletion kits, saving you time and money.

Different Sequencing Read Length

For good representation and even coverage of all transcripts in your experiment the library should have a size suitable for the chosen sequencing read length. The size of SENSE libraries for Illumina can be adjusted by simply modulating appropriate buffers during reverse transcription/ligation and purification steps.


SENSE mRNA-Seq Library Prep Kits are available for Illumina sequencing platforms.

Simple Multiplexing

SENSE libraries can be multiplexed with very well balanced barcode sequences, having 9,216 barcode options available for Illumina (single or dual indexing). Learn more about barcodes for multiplexing.


The SENSE kit features an all-in-one mRNA-Seq library preparation protocol. Most of the procedure is performed on magnetic beads, making it amenable to automation and decreasing purification time. The SENSE protocol has a simple workflow consisting of just 3 major steps.
Please find a schematic overview of the workflow for SENSE mRNA-Seq for Illumina below:

Poly(A) Selection
Step 1:
During the poly(A) selection step the total RNA samples are briefly heated
to resolve secondary structures and promote efficient hybridization.
Poly(A) Selection
Step 1:
The denatured total RNA is incubated with the oligodT beads, which
specifically bind polyadenylated RNAs. RNAs lacking a poly(A) tail are
then washed away, leaving only purified poly(A) RNA hybridized to the
Library Generation
Step 2:
As a result of this highly specific bead-based poly(A) selection step almost
all traces of cytoplasmic rRNA, tRNA, and other non-polyadenylated RNAs
are removed.
Library Generation
Step 2:
Library generation is based on Lexogen’s proprietary strand displacement
stop/ligation technology. The starter/stopper heterodimer mix is randomly
hybridized to the poly(A) RNA still bound to the magnetic beads.
Library Generation
Step 2:
A single-tube reverse transcription and ligation reaction extends the starter
to the next hybridized heterodimer, where the newly synthesized cDNA insert
is ligated to the stopper.
Library Generation
Step 2:
Distance is modulated by two different buffers resulting in different
conditions for the RT/ligation reaction
Library Generation
Step 2:
During second strand synthesis the RNA is hydrolyzed and the library is
converted to double-stranded DNA. The double-stranded library is purified
to remove magnetic beads and second strand synthesis reaction components.
Library Amplification
Step 3:
The library amplification is performed to add the complete adaptor
sequences required for cluster generation and to produce sufficient
material for quality control and sequencing. i7 and i5 indices can be
added during this step in order to be able to uniquely multiplex your
samples for the sequencing run.
Library Amplification
Step 3:
The finished library is purified from PCR components
which can interfere with quantification.

Library quantification can be performed with standard protocols, e.g., microcapillary electrophoresis or qPCR. Produced libraries are compatible with single-read or paired-end sequencing reagents.

Featured Publications


autoSENSE mRNA-Seq V2 for Illumina

autoSENSE V2 is the automated solution of the SENSE mRNA-Seq Library Prep Kit V2 for Illumina in combination with the software and the Automation Module, which is currently available for the Perkin Elmer Sciclone NGS Workstation in combination with the Zephyr Workstation for the post-PCR step. Please contact if you are interested in automating the SENSE protocol on any other liquid handler.

The main advantage of automating the RNA-Seq library preparation process is in reducing sample tracking errors while dramatically increasing throughput.

Fast and Fully Walk-Away Protocol

The whole autoSENSE V2 workflow is a complete walk-away protocol and 96 samples can be run within 9 hours, including about 2 hours of manual setup time. Since pre- and post-PCR can be run on separate machines the protocol time can be reduced by parallelizing the workflow.

Compatible with SENSE mRNA-Seq V2

The autoSENSE protocol is identical to the manual version of SENSE mRNA-Seq Library Prep Kit V2 and the software is downloadable from our website. With the Automation Module different library size selections can be performed and the workflow can be fully automated.

Flexibility of the Throughput

The autoSENSE kit is appropriate for preparing 9,216 barcoded libraries. The liquid handler program allows for processing of samples in multiples of 8 reactions (full columns of a 96-well plate). The reagents from a single kit can be distributed over several machine runs.

Avoiding Cross Contamination

Pre- and post PCR steps can be programmed on different machines to reduce the risk of cross-contamination of the pre-PCR samples by PCR products.


Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact

There are two major aspects of the SENSE technology contributing to its exceptional strand-specificity. Both of them are suppressing spurious second strand cDNA synthesis (see Figure 1), which introduces technical variation in detection of the antisense transcripts. Firstly, the protocol does not require RNA fragmentation as the insert size is determined by the distance between starter/stopper binding sites. Therefore, spurious second strand synthesis from the 5’ ends of RNA fragments is absent during reverse transcription. Secondly, when reverse transcriptase approaches the next hybridized primer during the first strand synthesis it is efficiently stopped, thus eliminating strand-displacement activity of the enzyme which can also cause spurious second strand synthesis.


Figure 1. Spurious second strand synthesis mechanisms. Firstly, when RT reaches 5’ ends it adds 1-5 nucleotides in a non template fashion, like a terminal transferase. When it happens the primer binding domain is free again and it can associate with a new primer, then RT flips back onto the first strand cDNA in a hairpin loop manner and a spurious second strand is initiated from this 5’ end. Secondly, when one random hexamer is extended it displaces at least to some degree the extension product of the second hexamer. This “free” displaced first strand can be further primed again with a random primer and thus create a spurious second strand.

The SENSE technology avoids both hairpin loop and strand displacement artifacts, providing the basis for the excellent strand-specificity.

ERCC spike-in controls are sets of artificial transcripts with known strand orientation which do not contain any antisense transcripts. Therefore all detected antisense ERCC reads can be considered false positives introduced during library preparation. In contrast, genome wide calculations of strandedness are conflated by true antisense transcription. Therefore true strand specificity can only be calculated on ERCC data, providing threshold levels for distinguishing endogenous antisense transcript levels from spurious second strand synthesis background.
SENSE mRNA-Seq: 8 ready-to-sequence libraries can be prepared in under 5 hours starting from total RNA.

autoSENSE mRNA-Seq: With autoSENSE 96 libraries can be prepared on a liquid handler within 9 hours, including ~2 hours preparation time and manual interventions.

Allow for more time when you carry out the protocol for the first time and perform QC (such as Bioanalyzer).

All SENSE mRNA-Seq kits already include the Poly(A) RNA Selection Kit (Cat. No. 039), all enzymes for reverse transcription, ligation, and PCR, as well as magnetic beads for purification and the barcodes for multiplexing.
You can also run the protocol without thermomixer. Just mix the reverse transcription and ligation reaction from time to time with a pipette. Alternatively you can also get the Poly(A) RNA Selection Kit (Cat. No. 039) in combination with the SENSE Total RNA-Seq kit which does not require a thermomixer.
We do not recommend using FFPE samples for SENSE mRNA-Seq. The protocol is performed on oligodT beads, hence the presence of a poly(A) tail of the RNA is essential. For optimal sequencing results we recommend using RNA with RIN score of 8 or greater. Libraries generated from low quality RNA may have a 3’ bias in sequencing results. However, SENSE Total RNA-Seq can be used on low quality samples (including FFPE).
SENSE mRNA-Seq: For preparation of the SENSE library the following equipment is needed (User-supplied Consumables and Equipment, SENSE User Guide (page 7)):
  • Magnetic rack
  • Benchtop centrifuge (12,000 x g, rotor compatible with 1.5 ml tubes or 96-well plates))
  • Calibrated single-channel pipettes for handling 1 µl to 1000 µl volumes
  • Thermomixer for 1.5 ml tubes (dry bath incubator with shaking function)
  • Thermocycler
  • UV spectrophotometer to quantify RNA
  • Recommended: Bioanalyzer (Agilent Technologies) for library quantification. Alternative quantification methods are: qPCR assays, Nanodrop or Qubit measurements

autoSENSE mRNA-Seq: For preparation of the SENSE library the following equipment is needed (User-supplied Consumables and Equipment, autoSENSE mRNA-Seq Library Prep Kit for Illumina on PE Sciclone/Zephyr User Guide (page 12)):

  • Magnetic rack (for manual bead wash)
  • Microplate centrifuge
  • Agencourt® 96-ring magnet (1 for Sciclone, 1 for Zephyr, PE: CLS 128316)
  • Magnet spacer (1 for Sciclone, PE: CLS 133514)
  • Inheco 96-well adapters (2 for Sciclone, 1 for Zephyr, PE: CLS 128372)
  • Inheco 96-well adapter/shaker (1 for Sciclone, 1 for Zephyr, PE: CLS 100852)
  • Thermocycler (Bio-Rad HSP-96 PCR-plate compatible)
  • UV-spectrophotometer to quantify RNA
  • Recommended: Automated microfluidic electrophoresis station Agilent Technologies 2100 Bioanalyzer, PerkinElmer LabChip GX II). Alternative quantification methods are: qPCR assays, Nanodrop or Qubit measurements
Based on our experience the best way to assess complexity is by generating discovery plots (Figure 2).


Figure 2. Discovery plots for 10 ng, 20 ng and 500 ng of input Universal Human Reference RNA (UHRR).

SENSE libraries have an excellent discovery rate on the transcript as well as the gene level.

The SENSE libraries are suitable for single- and paired-end sequencing. SENSE libraries generate R1 reads in a strand orientation opposite to the genomic reference. Read 2 generates sequences corresponding to the original RNA molecule. Please ensure the correct strand specificity in the corresponding parameters. For instance, we suggest to use TopHat or Cufflinks with the “–library.type” option “fr-firststrand”; for HTseq-count “–stranded=reverse” should be set.
For sequencing details we refer to Appendix I: Data Analysis, SENSE mRNA-Seq Library Prep Kit V2 User Guide (page 33).
A second peak between 1,000–9,000 bp (Fig. 3) is an indication of overcycling. The library prep has been very efficient and a lot of cDNA was generated. Hence, the PCR ran out of primers and template started to denature and reanneal improperly. This results in longer, bulky molecules that migrate at a lower speed on the Bioanalyzer chip or gels. This can interfere with exact library quantification if relying solely on the Bioanalyzer results. Therefore, a qPCR assay as recommended in Appendix C and D: qPCR and Reamplification, SENSE mRNA-Seq Library Prep Kit V2 for Illumina User Guide (p.25) for exact library quantification should be used additionally if such a high molecular weight peak occurs.

For future SENSE library preps on similar samples reduce your PCR cycle number accordingly to prevent overcycling. Overcycling may lead to a distortion in gene expression quantification and hence should be avoided.

Figure 3. Bioanalyzer traces of RTS (red) and RTL (blue) synthesized SENSE mRNA-Seq for Illumina libraries with a second peak in high molecular weight regions due to overcycling.

The low percentage of cytoplasmic ribosomal RNA demonstrates the exceptional specificity of the poly(A) selection in the SENSE protocol. Mitochondrial rRNAs, however, are polyadenylated and hence will also be selected when using the oligodT beads.

The number of cycles for your endpoint PCR depends on the type RNA (tissue, organism), the RNA quality and the RNA input amount. The reference values given in Appendix B, p.23 and Appendix C, p.25 are based on Universal Human Reference RNA input and the mRNA content of other RNA sources might differ. To be on the safe side and prevent under- or overcycling of your sample we recommend doing a qPCR first. Therefore each SENSE Kit contains 8 additional PCR reactions. For more reactions we offer a PCR Add-on Kit for Illumina (020.96) with 96 additional PCR reactions.

Dilute the samples you want to check by qPCR by adding 2 µl of Elution Buffer (EB) or RNase-free water to the 17 µl of your eluted library from step 33. For determining the cycle number of your endpoint PCR, please use 5 µl of the P7 Primer 7000 in step 37 of the protocol. Insert 1.7 µl (of the diluted 19 µl double-stranded library, step 21) into a qPCR reaction. Simply add SYBR Green I (or an equivalent fluorophore) to the PCR reaction to a final concentration of 0.1x (diluted in DMSO). Make the total reaction volume up to 30 µl with EB. Conduct at least 35 cycles to make sure the amplification reaches the plateau. Afterwards take the fluorescence value where the plateau is reached and calculate where the fluorescence is at 50 % of the maximum (see Fig. 4). The value where the fluorescence reaches the maximum (plateau) is taken (15388) and the fluorescence at 50 % of this values (7694) shows which cycle number is optimal for the endpoint PCRs. For the sample in Fig. 3 this would be 15 cycles when using 1/10th of your sample. If the optimal cycle number lies within two values, it is recommended to always round up to the higher number in order to get more yield. As in the endpoint PCR 10x more cDNA will be used compared to the qPCR, three cycles can be subtracted from the determined cycle number, hence in this example 12 cycles should be used for the endpoint PCR. This is the cycle number you should use for the endpoint PCR using the remaining 17 µl of the template. Once the number of cycles for the endpoint PCR is established for one type of sample, you can use it in the following experiments. For higher yields you can increase the fluorescence level of the endpoint PCR up to 80 % without overcycling your sample.


Figure 4: Calculation of the number of cycles for the endpoint PCR

Universal Human Reference RNA (UHRR, Agilent) is a good positive control, the most of the reference values given in the User Guide are also based on UHRR input.

SENSE mRNA-Seq and autoSENSE mRNA-Seq Library Prep Kits V2 (001.08, 001.24, 001.96) are appropriate for HiSeq 2000/2500, 3000, 4000, GAIIX, MiSeq, and NextSeq 500, 550 Illumina platforms.
Using 1 ng – 2 µg of total RNA input from various human, animal and plant tissues, fungi, and Universal Human Reference RNA yielded high quality libraries.
When using less than 50 ng of total RNA input please follow these recommendations.

1. Extend the primer hybridization in step 14 to 20 minutes.
2. Optional: Extend the time of the reverse transcription/ligation in step 16 to 2 hours for an increased yield.
3. Use 27 µl PB in step 39.

SENSE mRNA-Seq Library Prep Kit V2 is offered with two different reverse transcription and ligation mixes (RTS and RTL) to be used in step 12 of the library generation to adjust the library fragment size.

SENSE mRNA-Seq: RTS produces libraries with shorter mean insert sizes (265 bp), while RTL generates libraries with longer mean insert sizes (413 – 485 bp). SENSE mRNA-Seq Library Prep V2 for Illumina User Guide, Appendix C, Adjusting Library Size (p. 24).

autoSENSE mRNA-Seq: Without using the Automation Module (Cat. No. 024.96) RTS produces libraries with shorter mean insert sizes (208 bp), while RTL generates libraries with longer mean insert sizes (422 -545bp). For longer insert sizes the Automation Module is necessary.

The size of SENSE mRNA-Seq libraries can be adjusted to the desired sequencing length, yielding mean library sizes of around 250 – 500 bp. This is accomplished by modulating the insert range of the library generated during reverse transcription/ligation and by using different ratios in the magnetic bead purification. Please consult Appendix C: Adjusting Library Size, SENSE mRNA-Seq Library Prep V2 User Guide (page 24).
A 9 nt long random sequence of the starter and 6 nt long sequence of the stopper hybridize to the mRNA template. Trimming can be done with the same workflow for both reads in a paired-end dataset. Please ensure that the selected tool preserves the read-pair information. The first nine nucleotides need to be removed from Read 1 (starter side), while on the stopper side it is only six nucleotides (Read 2).

While trimming the first nucleotides introduced by the starter/stopper can decrease the number of reads of suitable length, the absolute number of mapping reads usually increases due to the improved read quality. Reads which are too short or have generally low quality scores should be removed from the set.

SENSE libraries can be multiplexed. i7 indices (i7 Index Plate, 7000-7096) are introduced during the PCR amplification step. The i5 Dual Indexing Add-on Kit (Cat. No. 047) allows for up to 384 different indexing combinations.
Multiplexing QuantSeq libraries with other library types in the same sequencing lane is not recommended. For further information please see FAQ 1.30.
In general, we recommend processing a minimum of 8 samples, and using a complete set of eight i7 indices for multiplexing (e.g., 7001-7008). However, if fewer indices are required care should be taken to always use indices which give a well-balanced signal in both lasers (red and green channels) for each nucleotide position. All columns (1 – 12) and rows (A – H) fulfill these criteria. An evaluation tool to check the color balance of index subsets is available under Support Tools. The individual libraries within a lane should be mixed at an equimolar ratio to ensure this balance.

Some examples for subsets of indices are listed below.

Two samples per lane: In step 37 use 5 µl of an equimolar mix of 7001-7004 for one sample and 5 µl of an equimolar mix of 7005-7008 for the second. Here four indices are applied to each sample in order to provide a perfect nucleotide balance of the index read-out. Alternatively, two indices can be applied per sample but check the color balance using the evaluation. For instance, use 2.5 µl of 7006 and 2.5 µl of 7008 for one sample and 2.5 µl of 7023 and 2.5 µl of 7096 for the second. Here two indices are applied to each sample in order to balance the red and green laser signals.

Four samples per lane: In step 37 use 5 µl of an equimolar mix of 7001-7002 for one sample, 5 µl of an equimolar mix of 7003-7004 for the second, 5 µl of an equimolar mix of 7005-7006for the third, and 5 µl of an equimolar mix of 7007-7008 for the fourth. Here two indices are applied to each sample in order to provide a perfect nucleotide balance of the index read-out. Alternatively, when using only 1 index per sample we recommend checking the color balance with the evaluation tool provided at Indices 7006, 7008, 7023, and 7096 are examples of four well-balanced indices.

Eight samples per lane: In step 37 use 5 µl of indices 7001-7008 (column 1 of the i7 Index Plate). Apply only one index to each sample.

Twelve samples per lane: In step 37 use indices 7001-7008 (column 1 of the i7 Index Plate) plus indices 7009-7012 (first 4 indices of column 2 of the i7 Index Plate). Apply only one index to each sample.

Final SENSE mRNA-Seq libraries contain platform-specific adapter sequences. For the sequencing the standard sequencing primers can be used.
The PCR Add-on Kit for Illumina (Cat. No. 020) includes a Reamplification Primer that can be used to add some PCR cycles on top of your undercycled libraries.
The Barcodes in the Barcode Plate (BC01-96) have been renamed to i7 Primer 7001-7096 in the i7 Index Plate and rearranged for a better nucleotide balance for sequencing when only few samples are multiplexed. With this new set-up all indices are now different from Illumina barcodes as the old BC05 was replaced with a new one.
Yes, the i5 Dual Indexing Add-on Kits can be used for dual indexing of SENSE mRNA libraries. Please refer to the i5 Dual Indexing Add-on Kits Instruction Manual (047IM109) for details of dual-indexed library amplification and purification.
For further information about the i5 Dual Indexing Add-on Kit for QuantSeq/SENSE please see the online frequently asked questions on the QuantSeq FWD product page.
We do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane. Though this is possible in principle, specific optimization of index combinations, library pooling conditions, and loading amounts may be required, even for advanced users. Sequencing complex pools that include different library types at different lane shares may have unpredictable effects on sequencing run metrics, read quality, read outputs, and/or demultiplexing performance. Lexogen assumes no responsibility for the altered performance of Lexogen libraries sequenced in combination with external library types in the same lane (or run).

Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq or SENSE libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).

You can do automated SENSE mRNA-Seq V2 already with SENSE mRNA-Seq Library Prep Library Prep V2 for Illumina after installation of the software package. If you want to have a fully walk-away protocol and select for larger library sizes the Automation Module for SENSE mRNA-Seq V2 is needed in addition. Please contact if you are interested in autoSENSE.
If oil is used for sealing the reaction wells the Phase 1-Pre PCR of the protocol does not require any operator intervention. Alternatively, a manual film sealing can be used.
The autoSENSE mRNA-Seq protocol is currently automated on PerkinElmer Sciclone NGS workstation. The Zephyr machine is used for the post-PCR purification, which could also be done manually or on any other liquid handler on which the PostPCR SPRI Purification application is established. Lexogen is planning to automate the SENSE protocols on all other liquid handling platforms used for NGS. Please contact us if you are interested in putting SENSE on your platform.
The robot protocol can be set up to run for any number of columns from 1 to 12 (simply by changing the respective entry in the Excel Workbook), but it will always take the barcodes from the first N wells of the P6 plate, starting by column 1. Therefore, you can run an assay with less than 96 (for example 24) reactions in multiples of 8, but you have to prepare a new P6 plate which will substitute the barcode plate contained in the kit. You can do this by simple transferring the reagents from the kit barcode plate to the columns 1, 2, and 3 (24 reactions = 3 columns x 8 rows) of a new HSP96 plate.
By selecting option YES, you can save about 15 minutes of each machine run time if you do the bead washing manually in a larger batch (takes about 10 minutes for several machine runs). Typically, manual prewashing only makes sense for high throughput sample processing.
The plate serves as a spacer to elevate the plates put on the B4 location. Because of presence of other consumables at the neighboring locations of B4, the tips on the robot head would otherwise not be able to plunge down to the bottom of the wells of a single HSP plate placed at B4.
Unfortunately no. The Phase1-PrePCR of the autoSENSE protocol is currently only available for the Sciclone NGS workstation.
The protocol is intended and programmed to run on this variant of the Sciclone liquid handler but this configuration has not been tested. Therefore, we recommend contacting Lexogen for on-site support with protocol installation.


SENSE mRNA-Seq Library Prep Kit V2 for Illumina

pdf User Guide for Illumina – update 17.07.2019
  Send us your publication & get the RNA T-shirt!

pdf PCR Add-on Kit for Illumina Instruction Manual – update 04.11.2020
pdf Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019
pdf SENSE Application Note
pdf Lexogen i7 and i5 Index Sequences – update 05.05.2020

 Library Quantification File

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