Qiagen QIAseq Stranded mRNA Library User Manual
Sample to Insight__
February 2021
QIAseq® Stranded
mRNA Library Kit
Handbook
For enrichment of polyadenylated RNA and
stranded RNAseq libraries for next-generation
sequencing using dual indexing
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Contents
Kit Contents ................................................................................................................ 3
Shipping and Storage ................................................................................................. 6
Intended Use .............................................................................................................. 6
Safety Information ....................................................................................................... 7
Quality Control ........................................................................................................... 7
Introduction ................................................................................................................ 8
Principle and procedure .................................................................................... 9
Equipment and Reagents to be Supplied by User .......................................................... 13
Important Notes ........................................................................................................ 14
Protocol: mRNA Enrichment ....................................................................................... 16
Protocol: Fragmentation/FastSelect RNA Removal ........................................................ 19
Protocol: First-strand Synthesis .................................................................................... 21
Protocol: Second-strand Synthesis, End-repair, and A-addition ........................................ 24
Protocol: Strand-specific Ligation ................................................................................. 27
Protocol: CleanStart Library Amplification .................................................................... 31
Recommendations: Library QC, Quantification, and Sequencing .................................... 35
Troubleshooting Guide .............................................................................................. 37
Appendix A: QIAseq Dual-Index Y-Adapters ................................................................ 39
Appendix B: Data Analysis Recommendations .............................................................. 43
Appendix C: mRNA Enrichment in 200 µl Plates .......................................................... 44
Appendix D: General Remarks on Handling RNA ...................................................... 46
Ordering Information ................................................................................................ 48
Document Revision History ......................................................................................... 52
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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Kit Contents
QIAseq Stranded mRNA Library Kit
Catalog no.
Number of reactions
Component
QIAseq Beads
QIAseq Beads N/A 10 ml 40 ml 40 ml 40 ml 40 ml
QIAseq Stranded mRNA Enrichment Kit
Pure mRNA Beads* Violet 2 x 600 µl 5 x 600 µl 5 x 600 µl 5 x 600 µl 5 x 600 µl
Buffer mRBB (binding buffer)† Clear 2 x 8 ml 4 x 8 ml 4 x 8 ml 4 x 8 ml 4 x 8 ml
Buffer OW2 (wash buffer) Clear 2 x 19 ml 7 x 19 ml 7 x 19 ml 7 x 19 ml 7 x 19 ml
RNase-Free Water Clear 1 x 10 ml 3 x 10 ml 3 x 10 ml 3 x 10 ml 3 x 10 ml
Buffer OEB (elution buffer) Clear 2 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml
Quick-Start Protocol N/A 1 1 1 1 1
QIAseq Stranded Total RNA Kit
RT Buffer, 5x Blue 1 x 215 µl 1 x 860 µl 1 x 860 µl 1 x 860 µl 1 x 860 µl
RT Enzyme Violet 1 x 28 µl 1 x 112 µl 1 x 112 µl 1 x 112 µl 1 x 112 µl
RNase Inhibitor Green 1 x 55 µl 1 x 212 µl 1 x 212 µl 1 x 212 µl 1 x 212 µl
Second Strand Buffer, 10x Blue 1 x 275 µl 1 x 1.1 ml 1 x 1.1 ml 1 x 1.1 ml 1 x 1.1 ml
Second Strand Enzyme Mix Violet 1 x 175 µl 1 x 700 µl 1 x 700 µl 1 x 700 µl 1 x 700 µl
DTT, 1 M Clear 1 x 1 ml 1 x 1 ml 1 x 1 ml 1 x 1 ml 1 x 1 ml
Ultralow Input Ligase Orange 2 x 65 µl 1 x 500 µl 1 x 500 µl 1 x 500 µl 1 x 500 µl
Ultralow Input Ligation Buffer, 4x Yellow 1 x 900 µl 3 x 900 µl 3 x 900 µl 3 x 900 µl 3 x 900 µl
CleanStart PCR Mix, 2x Red 1 x 660 µl 2 x 1.4 ml 2 x 1.4 ml 2 x 1.4 ml 2 x 1.4 ml
CleanStart PCR Primer Mix Clear 1 x 36 µl 1 x 150 µl 1 x 150 µl 1 x 150 µl 1 x 150 µl
Ligation Initiator Black 1 x 210 µl 1 x 840 µl 1 x 840 µl 1 x 840 µl 1 x 840 µl
QIAseq UDI Adapter Kit
QIAseq UDI Y-Adapter Plate (24) N/A 1 N/A N/A N/A N/A
QIAseq UDI Y-Adapter Kit A (96) N/A N/A 1 N/A N/A N/A
QIAseq UDI Y-Adapter Kit B (96) N/A N/A N/A 1 N/A N/A
QIAseq UDI Y-Adapter Kit C (96) N/A N/A N/A N/A 1 N/A
QIAseq UDI Y-Adapter Kit D (96) N/A N/A N/A N/A N/A 1
QIAseq Y-Adapter Reference Card N/A 1 1 1 1 1
Quick-Start Protocol N/A 3 3 3 3 3
* Caution: Pure mRNA Beads contain 0.1% sodium azide (NaN3) as a preservative. Dispose of azide-containing
solutions according to your institution’s waste-disposal guidelines. See “Safety Information”, page 7.
Tube cap color Volume
UDI (24)
180440
2424
UDI-A (96)
180441
9696
UDI-B (96)
180442
96
UDI-C (96)
180443
96
UDI-D (96)
180445
96
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
QIAseq Stranded mRNA Select Kit
Catalog no.
Number of reactions
Component Tube cap color Volume
QIAseq Beads
QIAseq Beads Clear 10 ml 40 ml
QIAseq Stranded mRNA Enrichment Kit
Pure mRNA Beads* Violet 2 x 600 µl 5 x 600 µl
Buffer mRBB (binding buffer)† Clear 2 x 8 ml 4 x 8 ml
Buffer OW2 (wash buffer) Clear 2 x 19 ml 7 x 19 ml
RNase-Free Water Clear 1 x 10 ml 3 x 10 ml
Buffer OEB (elution buffer) Clear 2 x 1.5 ml 5 x 1.5 ml
Quick-Start Protocol N/A 1 1
QIAseq Stranded RNA Lib Enzyme Kit
RT Buffer, 5x Blue 1 x 215 µl 1 x 860 µl
RT Enzyme Violet 1 x 28 µl 1 x 112 µl
RNase Inhibitor Green 1 x 55 µl 1 x 212 µl
Second Strand Buffer, 10x Blue 1 x 275 µl 1 x 1.1 ml
Second Strand Enzyme Mix Violet 1 x 175 µl 1 x 700 µl
DTT, 1 M Clear 1 x 1 ml 1 x 1 ml
Ultralow Input Ligase Orange 2 x 65 µl 1 x 500 µl
Ultralow Input Ligation Buffer, 4x Yellow 1 x 900 µl 3 x 900 µl
CleanStart PCR Mix, 2x Red 1 x 660 µl 2 x 1.4 ml
CleanStart PCR Primer Mix Clear 1 x 36 µl 1 x 150 µl
Ligation Initiator Black 1 x 210 µl 1 x 840 µl
QIAseq CDI Y-Adapter Kit
QIAseq CDI Y-Adapter Plate (24) N/A 1 N/A
QIAseq CD I Y-Adapter Plate (96) N/A N/A 1
QIAseq Y-Adapter Reference Card N/A 1 1
Quick-Start Protocol N/A 3 3
(24)
180773
24
(96)
180775
96
* Caution: Pure mRNA Beads contain 0.1% sodium azide (NaN3) as a preservative. Dispose of azide-containing
solutions according to your institution’s waste-disposal guidelines. See “Safety Information”, page 7.
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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The QIAseq Stranded Library Kits ship with a QIAseq Y-Adapter plate with either unique dualindex (UDI) adapters or combinatorial dual-index (CDI) adapters. To multiplex more than 96
libraries in a single sequencing run, simply combine the kits with different UDI Y-adapter plates.
For example, to multiplex 384 samples in a single flow cell line, prepare and combine libraries
from the QIAseq Stranded mRNA Library Kit UDI-A (96) with the QIAseq Stranded mRNA
Library Kit UDI-B (96), QIAseq Stranded mRNA Library Kit UDI-C (96), and QIAseq Stranded
mRNA Library Kit UDI-D (96). For more information on QIAseq Y-Adapter plates, please refer
to Appendix A, page 39.
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Shipping and Storage
The QIAseq Stranded mRNA Select Kit (cat. nos. 180773, 180775), QIAseq Stranded mRNA
Library Kit UDI (24) (cat. no. 180440), QIAseq Stranded mRNA Library Kit UDI-A (96) (cat.
no. 180441), QIAseq Stranded mRNA Library Kit UDI-B (96) (cat. no. 180442), QIAseq
Stranded mRNA Library Kit UDI-C (96) (cat. no. 180443), and QIAseq Stranded mRNA
Library Kit UDI-D (96) (cat. no. 180445) are shipped in 4 boxes.
In the QIAseq Stranded mRNA Enrichment Kit (cat. nos. 1105688, 1105689), the Buffers
mRBB and Pure mRNA Beads should be stored at 2–8°C (do not freeze). Buffer OW2 (wash
buffer), RNase-Free Water, and Buffer OEB (elution buffer) should be stored at room temperature
(15–25°C).
Store the QIAseq Stranded RNA Lib Enzyme Kit (cat. nos. 1122418, 1122419) at −30 to
−15°C.
Store the QIAseq UDI Y-Adapter Kit (cat. nos. 180312, 180314, 180316, 180318, 180310)
and QIAseq CDI Y-Adapter Kit (cat. nos. 180301, 180303) at −30 to −15°C.
The QIAseq Beads (cat. nos. 1107149, 1107460) should be stored at 2–8°C (do not freeze).
Important: Do not use expired beads as this will significantly reduce library yield
If stored under these conditions, the kit contents are stable until the date indicated on the box
labels.
.
Intended Use
The QIAseq Stranded mRNA Library Kit is intended for molecular biology applications. This
product is not intended for the diagnosis, prevention, or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We recommend
®
all users of QIAGEN
recombinant DNA experiments, or to other applicable guidelines.
products to adhere to the NIH guidelines that have been developed for
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit
and kit component.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of QIAseq
Stranded mRNA Library Kit is tested against predetermined specifications to ensure consistent
product quality.
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Introduction
A typical mammalian cell contains 10–30 pg of total RNA. The majority of RNA within cells
are rRNAs, with mRNA accounting for only 1–5% of the total cellular RNA. Approximately
360,000 mRNA molecules are present in a single mammalian cell, with approximately
12,000 different mRNA transcripts per cell. Some mRNAs comprise as much as 3% of the
mRNA pool, whereas others account for less than 0.01%. These “rare” or “low-abundance”
mRNAs may have a copy number of only 5–15 molecules per cell. However, these rare species
may account for as many as 11,000 different mRNA species, comprising 45% of the mRNA
population. For more information, see Alberts, B., et al. (1994)
3rd ed. New York: Garland Publishing, Inc.
In whole transcriptome next-generation sequencing (NGS) applications, it is of great interest
to maximize the amount of information generated from a single sequencing run. Enriching for
mRNA from the total cellular RNA pool allows scientists to reduce the amount of rRNA and
other non-mRNAs in their RNAseq data and increases the sequencing results from mRNA. The
QIAseq Stranded mRNA Enrichment Kit includes all the necessary reagents and buffers for the
isolation of poly(A)+ mRNA.
Molecular Biology of the Cell
.
NGS library preparation of RNA samples
The QIAseq Stranded mRNA Library Kits enable one-day, accurate stranded NGS library
construction from a broad range of RNA inputs. This kit includes magnetized QIAseq Beads
for fast and efficient reaction cleanups between protocol steps and Y-shaped sample index
adapter plates, which enable sample multiplexing. A total of 384 samples can be sequenced
together per lane on an Illumina NGS instrument by combining different sets of UDI sample
index plates.
Compared to other protocols, many novel advancements are included in the kit. During reverse
transcription, the optimized RT enzyme and buffers do not require the usage of toxic reagents
such as Actinomycin D to enhance strand specificity. In the second-strand synthesis reaction,
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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a specialized combination of enzymes and optimized buffering not only enables degradation
of the RNA strand, generation of a second cDNA strand, and generation of blunt DNA ends,
but it also guarantees the A-base addition required for the efficient ligation of Illuminacompatible adapters. The novel strand-specific ligation step establishes the strand specificity
of the QIAseq Stranded RNA Kit protocol without additional reagents or laborious and timeconsuming protocol steps. Finally, the CleanStart PCR Mix utilizes a high-fidelity DNA polymerase
to efficiently amplify the RNAseq library irrespective of GC content, while also using a novel method
to degrade previously generated QIAseq Stranded RNAseq libraries to guard against PCR amplicon
contamination.
Globin mRNA removal using QIAseq FastSelect RNA Removal Kits
When working with whole blood samples, globin mRNA represents a substantial library
contaminant. While mRNA enrichment effectively eliminates rRNA, globin mRNA remains a
concern. QIAseq FastSelect RNA Removal Kit (sold separately) is a breakthrough technology
that rapidly and efficiently removes up to 99% of globin mRNA during RNAseq library
preparation. Simply add the QIAseq FastSelect reagent during the NGS library preparation,
and unwanted RNAs are eliminated from the library.
Principle and procedure
The QIAseq Stranded mRNA Library Kit comprises the QIAseq Stranded mRNA Enrichment
Kit, the QIAseq Stranded RNA Lib Enzyme Kit, QIAseq Beads, and QIAseq Y-Adapter Kits.
The QIAseq FastSelect RNA Removal Kit (sold separately) is designed for fast and efficient
removal of globin mRNA during library preparation of whole blood samples. Together, these
kits enable preparation of mRNA-enriched, globin-mRNA–depleted, and strand-specific NGS
libraries from total RNA in less than 5.5 hours (Figure 1).
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Figure 1. QIAseq Stranded mRNA Library Kit workflow. The QIAseq Stranded mRNA Library Kit provides all necessary
reagents for mRNA enrichment and the preparation of strand-specific NGS libraries.
Optional: If you are working with total RNA from whole blood, the QIAseq FastSelect RNA Removal Kit provides all
necessary reagents for the removal of globin mRNA. The CleanStart library amplification step utilizes a high-fidelity
DNA polymerase to amplify the NGS libraries and prevent PCR contamination.
The following reactions are part of the workflow (Figure 2):
mRNA enrichment
Oligo-dT probes are covalently attached to the surface of the magnetically charged QIAGEN
Pure mRNA Beads. The mRNA binds rapidly and efficiently to the oligo-dT probes in the
presence of Buffer mRBB. The bound mRNA is then washed and eluted, providing a highly
enriched pool of mRNA.
RNA fragmentation/FastSelect RNA removal
Prior to RNA heat fragmentation, the FastSelect reagent is directly combined with the enriched
mRNA and the library prep-specific buffers. Heat fragmentation (if necessary) is then performed
and the reaction temperature is gradually cooled to room temperature (15–25°C).
First-strand synthesis
First-strand synthesis is performed using an RNase H- Reverse Transcriptase (RT) in combination
with random primers.
Second-strand synthesis, end-repair, and A-addition
Second-strand synthesis is performed using 5′ phosphorylated random primers. This enables
subsequent strand-specific ligation, as only one strand of the library is 5′ phosphorylated.
Strand-specific ligation
QIAseq adapters are efficiently asymmetrically ligated to the inserts due to the 5′ phosphate that
results from 5′ phosphorylated second-strand synthesis reaction.
CleanStart library amplification
QIAseq CleanStart PCR reagents use a proprietary PCR reaction, in conjunction with
modification enzymes, to ensure that previously constructed NGS libraries are removed.
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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Figure 2. QIAseq Stranded mRNA Library Kit with integrated QIAseq FastSelect globin mRNA removal.
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Next-generation sequencing
Libraries prepared with the QIAseq Stranded mRNA Library Kits can be sequenced with
Illumina
NGS systems (NextSeq® 500/550, HiSeq® 1000, HiSeq 1500, HiSeq 2000, HiSeq
2500, HiSeq 3000/4000, and NovaSeq™ 6000). When using unique dual indexes (cat.
nos. 180440, 180441, 180442, 180443, 180445), 74 bp paired-end reads and dual 10
bp index reads are required. When using combinatorial indexes (cat. no. 180773 or
180775), 76 bp paired-end reads and dual 8 bp index reads are required.
Data analysis
Downstream NGS data can be analyzed with the QIAGEN CLC Genomics Workbench. When
performing read alignment, the QIAseq Stranded Libraries represent the sense strand (or
positive DNA strand) of the RNA sequence due to the strand-specific ligation during the second
strand cDNA synthesis.
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Equipment and Reagents to be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs),
available from the product supplier.
100% ethanol (ACS grade)
Nuclease-free pipette tips and tubes
PCR tubes (0.2 ml individual tubes or tube strips) (VWR cat. no. 20170–012 or 93001–118) or
plates
1.5 ml LoBind
Ice
Microcentrifuge
Thermal cycler
Vortexer
Magnet for bead cleanups:
Tubes: MagneSphere
Plates: DynaMag™-96 Side Magnet (Thermo Fisher Scientific cat. no. 12331D)
Optional spike-in: ERCC RNA Spike-In Mix (Thermo Fisher Scientific cat. no. 4456740)
Library QC: 2100 Bioanalyzer
(Agilent cat. no. 5067-4626)
Preferred qPCR library quantification method: QIAseq Library Quant Array (cat. no. 333304) or
QIAseq Library Quant Assay Kit (cat. no. 333314)
®
tubes (Eppendorf cat. no. 022431021)
®
Technology Magnetic Separation Stand (Promega cat. no. Z5342)
®
(Agilent cat. no. G2939BA), Agilent High Sensitivity DNA Kit
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Important Notes
High-quality RNA is essential for robust library preparation and sequencing. QIAGEN provides
a range of solutions for purification of total RNA (Table 1).
Table 1. Recommended kits for purification of total RNA
Kit Cat. no. Starting material
RNeasy® Micro Kit 74004 Small amounts of cells and tissue
RNeasy Mini Kit 74104, 74106 Animal/human tissues and cells
RNeasy 96 Kit 74181, 74182 Animal/human tissues and cells
RNeasy FFPE Kit 73504 FFPE tissue samples
QIAamp® ccfDNA/RNA Kit 55184 Animal and human plasma and serum
exoRNeasy Midi Kit 77144 Animal and human plasma and serum
exoRNeasy Maxi Kit 77164 Animal and human plasma and serum
Ensure that total RNA samples are of high quality relative to their sample type. For additional
information, see “Appendix D: General Remarks on Handling RNA”.
RNA quantification: The concentration and purity of total RNA isolated from cells and
fresh/frozen tissues should be determined by measuring the absorbance in a
spectrophotometer, such as the QIAxpert
acids are highly dependent on pH, we recommend preparing dilutions and
measuring absorbance in 10 mM Tris·Cl, pH 7.5, instead of RNase-Free Water. Pure
RNA has an
RNA integrity: The integrity and size distribution of total RNA from cells and
A
:
A
ratio of 1.9–2.1 in 10 mM Tris·Cl, pH 7.5.
260
280
fresh/frozen tissue can be confirmed using an automated analysis system (such as the
QIAGEN QIAxcel or Agilent 2100 Bioanalyzer) that assesses RNA integrity by
monitoring the ribosomal RNA bands. Although the RNA integrity number (RIN)
should ideally be ≥8, successful library prep is still possible with samples with RIN
values <8. In situations where the RNA is highly degraded, we recommend to
consider using QIAseq FastSelect in combination with the QIAseq Stranded RNA
Library Kit UDI instead of using an mRNA enrichment strategy, if possible.
®
. Since the spectral properties of nucleic
QIAseq Stranded mRNA Library Kit Handbook 02/2021
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Indexing recommendations:
The QIAseq Stranded mRNA Library Kits include a QIAseq Y-Adapter plate with either CDI
adapters or UDI adapters. We recommend using the QIAseq Y-Adapter plates delivered with
the kit. Each QIAseq Stranded mRNA Library includes one of the following:
QIAseq Unique Dual-Index (UDI) Y-Adapter Plate (24)
QIAseq Unique Dual-Index (UDI) Y-Adapter Plate A, B, C, or D (96)
QIAseq Combinatorial Dual-Index (CDI) Y-Adapter Plate (24)
QIAseq Combinatorial Dual-Index (CDI) Y-Adapter Plate (96)
Sample multiplexing is one of the most important NGS tools for increasing throughput and
reducing costs. It works by combining multiple samples to be processed together in a single
sequencing run; as a consequence, sequencing reads need to be demultiplexed by reassigning
each single read to its original source library. This is facilitated by the integration of index
sequences into the individual adapter molecules.
CDI adapters use twelve i7 and eight i5 barcode motifs that can be combined to form up to 96
combinatory dual indices. In contrast, QIAseq UDI Adapters use a fixed combination of 2
unique barcode motifs per adapter molecule. Therefore, each single-index motif is only used
once on any UDI adapter plate. To multiplex more than 96 libraries in a single sequencing run,
combine kits with different UDI Y-adapter plates. Importantly, usage of UDI adapters effectively
mitigates the risk of read misassignment due to index hopping. This is enabled by filtering
misassigned reads during the demultiplexing of individual samples, thus generating highly
accurate output data. For this reason, usage of UDI adapters is highly recommended. For more
information on QIAseq Y-Adapter plates, please refer to “Appendix A: QIAseq Dual-Index Y-
Adapters”, page 39.
The protocol can be stopped at several steps and picked up on the following day. The stopping
points are as follows:
End of “Protocol: mRNA Enrichment “
End of “Protocol: First-strand Synthesis”
End of “Protocol: Second-strand Synthesis, End-repair, and A-addition”
End of “Protocol: Strand-specific Ligation”
End of “Protocol: CleanStart Library Amplification”
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QIAseq Stranded mRNA Library Kit Handbook 02/2021
Protocol: mRNA Enrichment
Important points before starting
This protocol is optimized for enriching RNA originating from all eukaryotic species with a poly-
A tail.
The recommended total RNA input is 100 ng – 1 µg.
See “Appendix C: mRNA Enrichment in 200 µl Plates” for enrichment of mRNA using 200 µl
strip tubes or 96-well plates.
Things to do before starting
All buffers and reagents should be vortexed before use to ensure thorough mixing.
Vortex the Pure mRNA Beads for 3 min before the first use or for 1 min before subsequent uses.
Heat a water bath or heating block to 70°C, and heat Buffer OEB to 70°C.
Unless otherwise indicated, all protocol steps, including centrifugation, should be performed at
room temperature.
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Procedure: mRNA enrichment in 1.5 ml tubes
1. Vortex Pure mRNA Beads for 1 min to thoroughly resuspend.
2. Prepare the enrichment reaction according to Table 2. Briefly centrifuge, vortex, and
centrifuge briefly again.
Table 2. Setup of enrichment reaction
Component Volume/reaction
Total RNA (100 ng – 1 µg) Variable
RNase Inhibitor 1 µl
Buffer mRBB 250 µl
Thoroughly resuspended Pure mRNA Beads 25 µl
Nuclease-Free Water Bring total reaction volume to 526 µl
Total volume 526 µl
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