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 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.

The protocol was extensively tested with input amounts of 1 ng to 50 ng µg of total RNA input using RNA extracted from mouse liver, kidney, brain, lung, spleen, thymus, and heart as well as Universal Human ERCC spike-in controls, a set of artificial transcripts with known strand orientation which do not contain any antisense transcripts. Therefore, all detected antisense ERCC reads can be considered as false positives introduced during library preparation. In contrast, genome wide calculations of strand-specificity 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.

8 ready-to-sequence SENSE Total RNA-Seq libraries can be prepared within 3.5 hours starting from poly(A) or rRNA depleted total RNA. Allow for an additional 2 h for the rRNA depletion of total RNA using RiboCop or 1 hour for the poly(A) selection with the Poly(A) RNA Selection Kit, respectively.
Allow for more time when you carry out the protocol for the first time and QC (such as Bioanalyzer) if needed.

The SENSE Total RNA-Seq kit already includes external barcodes, all enzymes for reverse transcription, ligation, and PCR, as well as all reagents for purification. It is also available as a bundle with the RiboCop rRNA Depletion Kit (Cat. No. 042).
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.

Typical inputs of poly(A) selected and rRNA depleted RNA from various tissues, plants, bacteria of 0.5 ng to 50 ng RNA will generate high quality libraries.

SENSE Total RNA-Seq is suitable for FFPE and low quality RNA samples, please refer to the respective User Guide.

For preparation of the SENSE Total RNA-Seq libraries the following equipment is needed (User-supplied Consumables and Equipment, SENSE Total RNA-Seq Library Prep Kit User Guide (page 8)):

• 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

SENSE Total RNA-Seq library prep kits (Cat. No. 009 and 042) are appropriate only for Illumina platforms (HiSeq2000, HiSeq3000, HiSeq4000, GAIIX, MiSeq, NextSeq 500, NextSeq 550).

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 H: Sequencing, SENSE Total RNA-Seq Library Prep Kit User Guide (page 32).

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.

The number of cycles for your endpoint PCR depends on the type of the RNA (tissue, organism), the RNA quality and the RNA input amount. The reference values given in Appendix B, p.21 and Appendix D, p.24 are based on RiboCop rRNA depleted Universal Human Reference RNA input and the RNA 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 21. For determining the cycle number of your endpoint PCR, please use 5 µl of the P7 Primer 7000 in step 25 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 1/10^th 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 3: Calculation of the number of cycles for the endpoint PCR

With the SENSE Total RNA-Seq kit you can choose between four different library sizes based on your sequencing needs when using intact or partially degraded RNA. Depending on the combination of PS and BD used in step 10 (SENSE Total RNA-Seq Library Prep Kit User Guide (p. 13), mean library sizes between 370 – 510 bp will be produced, resulting in library fragments of 150 – 1500 bp. For highly degraded or FFPE samples the final library size is quite dependent on the level of degradation, but will be around 150 bp.

The size of SENSE Total RNA-Seq libraries can be adjusted to the desired sequencing length, yielding mean library sizes between 370 – 510 bp. This is accomplished by modulating the insert range of the library generated after second strand synthesis by different size cut offs during magnetic bead purification. Please consult Appendix E: Adjusting Library Size, SENSE Total RNA-Seq Library Prep Kit User Guide (p. 27). For highly degraded or FFPE samples the final library size depends on the level of degradation and cannot easily be adjusted. The insert size will be around 150 bp and shorter sequencing lengths are recommended.

SENSE Total RNA-Seq uses a 9 nt long random sequence of the starter and a 6 nt long random sequence of the stopper hybridized to the RNA template. Therefore, the first nine nucleotides need to be removed from Read 1 and the first six nucleotides from Read 2.

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 using four additional i5 indices.
Multiplexing QuantSeq libraries with other library types in the same sequencing lane is not recommended. For further information please see FAQ 1.30.

Final SENSE Total RNA-Seq libraries contain Illumina-specific adapter sequences. For the sequencing the standard primers can be used.

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.

The PCR Add-on Kit for Illumina includes a Reamplification Primer that can be used to add some PCR cycles on top of your single indexed (i5) undercycled libraries, consult Appendix D: Library Reamplification, SENSE Total RNA-Seq Library Prep Kit User Guide (p. 26). For reamplification of dual indexed (i7 and i5) libraries contact info@lexogen.com

Please refer to the dedicated User Guide for FFPE Total RNA-Seq Library Prep. FFPE RNA is often heavily fragmented, hence the starter/stopper heterodimers will hybridize in shorter distances, generating small (or too small) insert sizes. Therefore the primers need to be diluted and the purification steps and PCR cycles need to be adjusted.

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 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.

Yes, the i5 Dual Indexing Add-on Kits can be used for dual indexing of SENSE Total 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).