Description

SENSE Total RNA-Seq Library Prep Kit

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. Four additional i5 Index Primer available in the i5 Dual Indexing Add-on Kit (Cat. No. 047) allow for an even higher degree of unique indexing and multiplexing of up to 384 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

NGS-ready libraries can be produced from poly(A) selected or rRNA depleted RNA samples in under 3.5 hours with less than 50 % hands-on time, allowing RNA extraction, library preparation, and quality control to be performed in one day.

Flexible Input Requirements

The typical input amount of poly(A) or rRNA depleted RNA is 0.5 ng – 50 ng. SENSE Total library generation can furthermore be done on intact as well as highly degraded RNA samples (including FFPE).

Libraries for 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 can be adjusted by simply modulating the purification steps.

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.

Library Generation
Step 1:
The starting material is either rRNA depleted or poly(A) selected RNA.
Library Generation
Step 1:
Library generation starts with the random hybridization of the starter/stopper
heterodimer mix to the poly(A) selected or rRNA depleted RNA.
Library Generation
Step 1:
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 1:
The insert size is dictated by the distance between
starter/stopper binding sites.
Library Generation
Step 1:
During second strand synthesis the RNA is hydrolyzed and the library is
converted to double-stranded DNA. The double-stranded library is purified
using magnetic beads to adjust the library length and get rid of second
strand synthesis reaction components.
Library Amplification
Step 2:
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 2:
The finished library is purified from PCR components which
can interfere with quantification.

For viewing the whole workflow on page please click here

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.

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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.

Spurious-SS-Synthesis-Mechanisms
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.

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 G: Sequencing, SENSE Total RNA-Seq Library Prep Kit User Guide (page 29).

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: qPCR and Reamplification, SENSE Total RNA-Seq Library Prep Kit for Illumina User Guide (p.23) 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.

Overcycled_SENSE_Libraries

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). Conduct at least 30 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.

Calculation_of_the_number_of_cycle_for_the_endpoint_PCR

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 D: Adjusting Library Size, SENSE Total RNA-Seq Library Prep Kit User Guide (p. 24). 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.
SENSE libraries can be easily multiplexed with samples from other library preps, as the i7 Index Plate contains different barcodes than the standard Illumina ones, consult Appendix F: Multiplexing, SENSE Total RNA-Seq Library Prep for Illumina User Guide (page 27).
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 C: qPCR and Reamplification, SENSE Total RNA-Seq Library Prep Kit User Guide (p. 23). 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 at this link. 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.

Downloads

SENSE Total RNA-Seq Library Prep Kit for Illumina

pdf User Guide for intact RNA or partially degraded RNA – update 17.02.2017 (Introduction of new i7 Index Plate; referral to i5 Dual Indexing Add-on Kit; qPCR endpoint determination using only 1.7 µl template and set to 50 % FU)
pdf User Guide for heavily degraded or FFPE samples – update 17.02.2017 (Introduction of new i7 Index Plate; referral to i5 Dual Indexing Add-on Kit; qPCR endpoint determination using only 1.7 µl template and set to 50 % FU)
pdf PCR Add-on Kit for Illumina Instruction Manual – update 17.02.2017 (Renaming of BC00 to i7 Primer 7000; qPCR endpoint determination using only 1.7 µl template and set to 50 % FU)
pdf i5 Dual Indexing Add-on Kit for QuantSeq/SENSE Instruction Manual – update 17.02.2017 (Inclusion of QuantSeq-Flex; Renaming of i7 indices in i7 Index Plate)
pdf Product Flyer
pdf SENSE Total RNA-Seq for Illumina Index Primer Overview (i7 Index Plate) – for kits bought after 17.02.2017
pdf Barcode Plate Overview – for kits bought before 17.02.2017

Material Safety Datasheets

pdf 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.

SENSE Bioinformatics Data Analysis

Find more about the SENSE Data Analysis here.

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