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.
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:
to resolve secondary structures and promote efficient hybridization.
specifically bind polyadenylated RNAs. RNAs lacking a poly(A) tail are
then washed away, leaving only purified poly(A) RNA hybridized to the
all traces of cytoplasmic rRNA, tRNA, and other non-polyadenylated RNAs
stop/ligation technology. The starter/stopper heterodimer mix is randomly
hybridized to the poly(A) RNA still bound to the magnetic beads.
to the next hybridized heterodimer, where the newly synthesized cDNA insert
is ligated to the stopper.
conditions for the RT/ligation reaction
converted to double-stranded DNA. The double-stranded library is purified
to remove magnetic beads and second strand synthesis reaction components.
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.
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.
For viewing the whole workflow on page please click here
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 email@example.com 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.
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 firstname.lastname@example.org.
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.
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).
- 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)
- 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
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.
For sequencing details we refer to Appendix H: Data Analysis, SENSE mRNA-Seq Library Prep Kit V2 User Guide (page 30).
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 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.22 and Appendix C, p.23 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). 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.
Figure 4: Calculation of the number of cycles for the endpoint PCR
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. 23).
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.
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 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 mRNA-Seq Library Prep V2 for Illumina User Guide (page 27).
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.
SENSE mRNA-Seq Library Prep Kit V2 for Illumina
User Guide for Illumina – 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)
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)
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)
SENSE Application Note
SENSE mRNA-Seq for Illumina Index Primer Overview (i7 Index Plate) – for kits bought after 17.02.2017
Barcode Plate Overview – for kits bought before 17.02.2017
SENSE mRNA-Seq Library Prep Kit for Ion Torrent
Material Safety Datasheets
SENSE Bioinformatics Data Analysis
Find more about the SENSE Data Analysis here.