Dear Customers,
SENSE Total RNA-Seq Library Prep Kit is discontinued since 1.1.2020. We recommend instead to use our CORALL Total RNA-Seq Library Prep Kit.
Description
SENSE Total RNA-Seq Library Prep Kit (discontinued)
SENSE Total RNA-Seq is a strand-specific library prep kit for accurate gene expression profiling, whole 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 by omitting reverse transcription artifacts, and therefore avoids spurious second strand cDNA synthesis, which may result in the detection of false antisense transcription. Thereby an exceptional (>99.9 %) strand-specificity and reduced experimental noise is reached, enabling the detection and quantification of antisense transcripts with high confidence.
Simple Multiplexing
With the up to 96 i7 Index Primer included in the kit libraries can be easily multiplexed. 96 additional i5 Index Primers available in the Lexogen i5 6 nt Dual Indexing Add-on Kit (Cat. No. 047) allow for an even higher degree of unique indexing and multiplexing of up to 9,216 samples.
Streamlined Ribosomal RNA Depletion
For efficient utilization of RNA-Seq reads highly abundant ribosomal RNA should be eliminated prior library preparation. SENSE Total RNA-Seq Kit is available in combination with RiboCop rRNA Depletion Kit. This is a complete solution for RNA-Seq library preparation, starting with total RNA and finishing with a ready-to-sequence library.
Rapid Turnaround
Flexible Input Requirements
Libraries for Different Sequencing Read Length
Workflow
The SENSE Total RNA-Seq kit is based on Lexogen’s proprietary strand-displacement stop/ligation technology and needs poly(A) selected or rRNA depleted RNA input.
For viewing the whole workflow on page please click here
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FAQ
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 support@lexogen.com.
Figure 1: Spurious second strand synthesis mechanisms. Firstly, when reverse transcriptase 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 freed again and this can associate with a new primer, then reverse transcriptase flips back onto the first strand cDNA in a hairpin loop manner and a spurious second strand synthesis 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.
Allow for more time when you carry out the protocol for the first time and QC (such as Bioanalyzer) if needed.
Magnetic beads for poly(A) selection using the Poly(A) RNA Selection kit need to be purchased separately. However, if mRNA sequencing is of interest, we would recommend using the SENSE mRNA-Seq Library Prep Kit which already includes poly(A) selection.
- Benchtop centrifuge (12,000 x g, rotor compatible with 1.5 ml tubes)
- Magnetic rack or plate
- Calibrated single-channel pipettes for handling 1 µl to 1000 µl volumes
- Thermocycler
- UV spectrophotometer to quantify RNA
- Recommended: Bioanalyzer (Agilent Technologies) for library quantification. Alternative quantification methods are: qPCR assays, Nanodrop or Qubit measurements
A second peak between 1,000–9,000 bp (Fig. 2) 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 Appendix D: qPCR and Library Reamplification, SENSE Total RNA-Seq Library Prep Kit 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 2: Bioanalyzer traces of RTS (red) and RTL (blue) synthesized SENSE for Illumina libraries with a second peak in high molecular weight regions due to overcycling.
Figure 3: Calculation of the number of cycles for the endpoint PCR
Multiplexing QuantSeq libraries with other library types in the same sequencing lane is not recommended. For further information please see FAQ 1.30.
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 www.lexogen.com. 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.
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.
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).
Downloads
SENSE Total RNA-Seq Library Prep Kit for Illumina
User Guide for intact RNA or partially degraded RNA – update 10.10.2018
User Guide for heavily degraded or FFPE samples – update 10.10.2018
PCR Add-on Kit for Illumina Instruction Manual – update 04.11.2020
Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019
Product Flyer
Lexogen i7 and i5 Index Sequences – including for kits bought before 17.02.2017
Library Quantification Calculation File
Material Safety Datasheets
MSDS information for SENSE Total RNA-Seq Library Prep Kit for Illumina
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.