KAPA Stranded mRNA-Seq Kits

Even difficult messages should be understood.

KAPA Stranded mRNA-Seq Kits include all the enzymes and buffers required for cDNA library preparation for Illumina Next-Generation Sequencing, utilizing 100 ng – 4 µg of total RNA. KAPA mRNA Capture Beads are included for isolation of poly(A)-tailed RNA. Kits provide precise measurement of strand orientation (>99%), uniform coverage, and high-confidence mapping of alternate transcripts, and are optimized for the improved coverage of GC-rich and low-abundance transcripts.* Kits contain KAPA HiFi for high-efficiency and low-bias library amplification, as well as KAPA mRNA Capture Beads and a streamlined, “with-bead” protocol.

KAPA Stranded RNA-Seq Kits include all the enzymes and buffers required for cDNA library preparation for Illumina Next-Generation Sequencing, but do not contain the KAPA mRNA Capture Beads. Kits can be used to prepare libraries from 10 – 400 ng of either poly(A)-selected, ribosomally depleted, or total RNA.

NEW! KAPA Dual-Indexed Adapter Kits are now available. For more information on KAPA Adapter Kits, scroll down to the Ordering section, or download the KAPA Adapter and Bead Calculator.

Download Adapter and Bead Calculator

 

*Data on file.
For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Improved coverage of GC-rich transcripts. The 5' and 3' exons (outlined in red) of the DVL3 transcript contain regions of very high GC content. These regions are covered to a significantly greater depth by the KAPA Stranded mRNA-Seq Kit (green) in comparison to the Illumina® TruSeq™ Stranded mRNA Sample Prep Kit (orange). Data on file.

Uncover challenging transcripts 

  • Improved coverage of GC-rich transcripts
  • Enhanced identification of exonic regions
Improved coverage of low-abundance transcripts. GLTPD1, a lesser-expressed transcript, is covered more comprehensively with the KAPA Stranded mRNA-Seq Kit (green) at 500 ng input total RNA (outlined in red). In comparison, Illumina TruSeq Stranded mRNA Sample Prep Kit (orange) shows coverage gaps, even with higher inputs (4 μg). Data on file.

Detect low-abundance transcripts

  • Enables identification of transcripts missed by competitor kits, even with high input*
  • High uniformity across varying amounts of sample input*
Highly mapped reads and strand specificity enable sensitive detection of expressed genes. With similar numbers of filter-passed reads (>30M), KAPA Stranded mRNA-Seq libraries generate a greater percentage of mapped reads and lower duplication rates than analogous libraries prepared with the Illumina Stranded mRNA Sample Prep Kit while maintaining 99% strand specificity. Data on file.

Identify more genes

  • Higher percentage of uniquely mapped reads compared to Illumina TruSeq™ Stranded mRNA Sample Prep Kits*
  • Lower duplication rates yield better coverage
Minimal positional bias. With intact, high-quality RNA input, 3' positional coverage bias was minimized (average across top 1000 transcripts shown). Compared to the Illumina TruSeq Stranded mRNA Sample Prep Kit (orange), KAPA Stranded mRNA-Seq Kit (green) produces equivalent 5’ – 3’ coverage across transcript. Data on file.

Maintain high coverage uniformity

  • Minimal 5’–3′ bias across transcripts
  • More uniform distribution of reads over each transcript

 

*Data on file.

Related Products

Sequencing challenging RNA samples? How about DNA? Check out these Kapa NGS products to improve your workflow and results:

Sample QC

KAPA hgDNA Quantification and QC Kit

Library Amplification

KAPA Library Amplification Kits

Library Preparation

KAPA Hyper Prep Kits

Library Quantification

KAPA Library Quantification Kits

Applications
  • Gene expression
  • Single nucleotide variation (SNV) discovery
  • Post-transcriptional SNVs
  • Fusion gene identification
  • Targeted transcriptome
  • Whole transcriptome
Kit Specifications and Contents / Storage

Kits can be stored for up to 8 months at -20˚C. KAPA mRNA Capture Beads can be stored for up to 8 months at -4˚C.

Components

mRNA_Component Chart

Specifications

Spec
Description
Compatible Platform
Illumina HiSeq, NextSeq, MiSeq and GAIIx
Starting Material
Total RNA, mRNA, or ribosomal depleted RNA
Input Amount
Stranded mRNA-Seq Kits: 100 ng–4 µg Stranded RNA-Seq Kits: 10 ng–400 ng
FAQs

KAPA Stranded mRNA-Seq Kits:

Please see below for KAPA Stranded RNA-Seq Kit Frequently Asked Questions.

Are these kits compatible with small RNA library preparation?

No, these kits are not compatible with small RNA.

What are the input requirements for the KAPA Stranded mRNA-Seq Kits?

100 ng–4 µg of purified total RNA, dissolved in <50 µL RNase-free water.

What are the major steps in library construction?

  • mRNA capture using magnetic oligo-dT beads
  • Fragmentation using heat and magnesium
  • 1st Strand cDNA Synthesis using random priming
  • 2nd Strand cDNA Synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA) and incorporates dUTP in the second cDNA strand
  • A-tailing to add dAMP to the 3′-ends of the dscDNA library fragments
  • Adapter ligation, where dsDNA adapters with 3′-dTMP overhangs are ligated to A-tailed library insert fragments
  • Library amplification to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR; the strand marked with dUTP is not amplified.

Do the kits provide polyA capture beads?

Yes, the KAPA Stranded mRNA-Seq Kits include the KAPA mRNA capture beads required for poly(A) mRNA capture.

Do the kits offer strand-specific information?

Yes, during 2nd strand synthesis, the DNA:RNA hybrid is converted to double-stranded DNA, with dUTP incorporated into the second cDNA strand. During library amplification the strand containing dUTP is not amplified, allowing strand-specific sequencing. This kit retains accurate strand origin information in ˃99% of unique mapped reads.

Are there safe stopping points in the sample preparation process?

The library construction process from RNA fragmentation through library amplification can be performed in 6-8 hours, depending on the number of samples being processed, and experience. If necessary, the protocol may be paused safely after any of the following steps:

  • After mRNA capture, resuspend the beads in 22 µL of Fragment, Prime, and Elute Buffer, and store the sealed tube at 4°C for up to 24 hours.
  • After the 2nd strand synthesis cleanup, resuspend the washed beads in 15 µL of 1x A-Tailing Buffer (without enzyme) and store the sealed tube at 4°C for up to 24 hours.
  • After the first post-ligation cleanup, store the resuspended beads at 4°C for up to 24 hours. Do not freeze the beads, as this can result in dramatic loss of DNA.
  • After the second post-ligation cleanup, store the eluted, unamplified library DNA at 4°C for up to 24 hours, or at -20°C for up to 1 week.

How long can adapter-ligated cDNA be stored?

Purified, adapter-ligated cDNA can be stored at 4°C for one week or at -20°C for at least one month, before amplification and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0) and minimize the number of freeze-thaw cycles.

What adapters can I use with this kit and at what concentration?

KAPA Adapters are recommended for use with KAPA Stranded mRNA-Seq Kits. Kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in  SeqCap EZ, Illumina TruSeq, Agilent SureSelect, and other similar library construction and target capture workflows. Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency.

While it is not necessary to adjust adapter concentrations to accommodate moderate sample-to-sample variation, an adapter concentration appropriate for the amount of input RNA is recommended. The table below summarizes recommended adapter concentrations for various inputs into the mRNA capture reaction.

Quantity of starting material Adapter stock concentration Adapter concentration in ligation reaction
2001 – 4000 ng 1400 nM 100 nM
501 – 2000 ng 700 nM 50 nM
251 – 500 ng 350 nM 25 nM
100 – 250 ng 140 nM 10 nM

Where do I find more information about KAPA Adapters?

Please refer to the KAPA Dual-Indexed and Single-Indexed Adapter Technical Data Sheet for information about barcode sequences, pooling, kit configurations, formulation, and dilution for different KAPA DNA and RNA library preparation kits and inputs.

What QC testing is performed on KAPA Adapters?

KAPA Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:

  • optimal library construction efficiency
  •  minimal levels of adapter-dimer formation
  • nominal levels of barcode cross-contamination

Library construction efficiency and adapter-dimer formatin are assessed in a low-input library construction workflow. The conversion rate achieved in the assay indicates library construction efficiency. This is calculated by measuring the yield of adapter-ligated library (before any amplification) by qPCR (using the KAPA Library Quantification Kit), and expressing this as a % of input DNA. To assess adapter-dimer formation, a modified library construction protocol— designed to measure adapter dimer with high sensitivity—is used.

Barcode cross-contamination is assessed by sequencing. Each adapter is ligated to a unique, synthetic insert of known sequence, using a standard library construction protocol. These constructs pooled and sequenced on a MiSeq. For every barcode, the number of reads (in the range of 115,000 – 500,000) associated with each insert is counted, and the total % correct inserts calculated. Contamination of any barcode with any other single barcode is guaranteed to be <0.25%. The total level of contamination for any barcode is typically in the range of 0.1 – 0.5%. This assay is unable to distinguish between chemical cross-contamination and adapter “cross-talk”, and measures the total number of incorrect inserts resulting from both phenomena.

What is the fragmentation protocol with this kit?

RNA is fragmented using high temperature in the presence of magnesium. Depending on the origin and integrity of the input RNA, and the intended application, different RNA fragmentation protocols are provided to obtain the required insert size distribution. For intact RNA such as that extracted from fresh/frozen tissue, longer fragmentation is required at higher temperatures. For degraded or fragmented RNA (e.g. from older samples or formalin-fixed-paraffin-embedded (FFPE) tissue), use a lower temperature and/or shorter times. The table below outlines various fragmentation parameters depending on the input RNA and the desired insert size.

Desired Insert Size Temperature Duration
100-200 bp 94˚C 8 min
200-300 bp 94˚C 6 min
300-400 bp 85˚C 6 min

How should I measure the final library?

The size distribution of the double-stranded cDNA and/or final amplified library should be confirmed with an electrophoretic method. The quantification of the library should be done with a qPCR based quantification kit such as the KAPA Library Quantification Kits for Illumina platforms. These kits employ primers based on the Illumina flow cell oligos, and can be used to quantify libraries that are ready for flow-cell amplification.

Which polymerase is used for amplification?

KAPA HiFi HotStart is the enzyme provided in the KAPA HiFi HotStart ReadyMix. This is a novel B-family DNA polymerase engineered for low-bias, high fidelity PCR and is the reagent of choice for NGS library amplification1,2,3.

  1. Oyola, S.O. et al. BMC Genomics 13, 1 (2012).
  2. Quail M.A. et al. Nature Methods 9, 10-11 (2012).
  3. Quail M.A. et al. BMC Genomics 13, 341 (2012).

How many cycles should I use when amplifying my adapter-ligated library?

To minimize over-amplification and associated unwanted artifacts, the number of PCR cycles should be optimized to produce a final amplified library with a concentration range of 10-30 ng/µL which is equivalent to 0.5-1.5 µg of DNA per 50 µL reaction. The number of cycles recommended below should be used as a guide for library amplification, but cycle numbers may have to be adjusted (± 4 cycles) depending on library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

Input RNA Number of Cycles
100–250 ng 10–16
251–500 ng 10–14
501–2000 ng 8–12
2001–4000 ng 6–10

What are the storage conditions for this kit?

KAPA Stranded mRNA-Seq Kits are supplied in two boxes. Box 1 contains capture beads and buffers, and is shipped at 4˚C. The contents of Box 1 must not be frozen, as this will damage beads. Upon receipt, store Box 1 at 4˚C. Box 2 contains enzymes and buffers for cDNA synthesis and library preparation, and is shipped on dry ice or ice packs, depending on the destination country. The contents of Box 2 are temperature-sensitive, and appropriate care should be taken during storage. Upon receipt, store Box 2 at -20˚C in a constant temperature freezer. The 1st Strand Synthesis Buffer and PEG/NaCl Solution supplied in Box 2 are light-sensitive, and should be protected from light during storage. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.

 

KAPA Stranded RNA-Seq Kits:

Are these kits compatible with small RNA library preparation?

No, these kits are not compatible with small RNA.

What are the input requirements of the KAPA Stranded RNA-Seq Kits?

10 – 400 ng of purified RNA (e.g. mRNA captured, rRNA-depleted, or total RNA), dissolved in ≤10 µL of water.

What are the major steps in library construction?

  • Fragmentation using heat and magnesium
  • 1st Strand cDNA Synthesis using random priming
  • 2nd Strand cDNA Synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA) and incorporates dUTP in the second cDNA strand.
  • A-tailing to add dAMP to the 3′-ends of the dscDNA library fragments
  • Adapter ligation, where dsDNA adapters with 3′-dTMP overhangs are ligated to A-tailed library insert fragments.
  • Library amplification to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR. The strand marked with dUTP is not amplified.

Are poly(A) capture beads included in the kits?

No, the KAPA Stranded RNA-Seq Kits do not include beads for mRNA capture. We recommend the KAPA Stranded mRNA-Seq Kits, which include the KAPA mRNA Capture Beads for mRNA enrichment.

Do the kits offer strand-specific information?

Yes, during 2nd strand synthesis, the DNA:RNA hybrid is converted to double-stranded DNA, with dUTP incorporated into the second cDNA strand. During library amplification the strand containing dUTP is not amplified, allowing strand-specific sequencing. This kit retains accurate strand origin information in ˃99% of unique mapped reads.

Are there safe stopping points in the sample preparation process?

The library construction process from RNA fragmentation through library amplification can be performed in 6-8 hours, depending on the number of samples being processed, and experience. If necessary, the protocol may be paused safely after any of the following steps:

  • After the 2nd strand synthesis cleanup, resuspend the washed beads in 15 µL of 1x A-Tailing Buffer (without enzyme) and store the sealed tube at 4°C for up to 24 hours
  • After the first post-ligation cleanup, store the resuspended beads at 4°C for up to 24 hours. Do not freeze the beads, as this can result in dramatic loss of DNA.
  • After the second post-ligation cleanup, store the eluted, unamplified library DNA at 4°C for up to 24 hours, or at -20°C for up to 1 week.

How long can adapter-ligated cDNA be stored?

Purified, adapter-ligated cDNA can be stored at 4°C for one week or at -20°C for at least one month, before amplification and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0) and minimize the number of freeze-thaw cycles.

What adapters can I use with this kit and at what concentration?

KAPA Adapters are recommended for use with KAPA Stranded RNA-Seq Kits. However, kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in Illumina TruSe™ Roche NimbleGen SeqCap EZ, Agilent SureSelect, and other similar library construction and target capture workflows. Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency.

Quantity of starting material Adapter stock concentration Adapter concentration in ligation reaction
201 – 400 ng 1400 nM 100 nM
51-200 ng 700 nM 50 nM
10 – 50 ng 350 nM 25 nM

Low Concentration (1.5 µM) KAPA Adapter Kits are recommended for all inputs. For assistance with adapter compatibility and ordering, please visit kapabiosystems.com/support.

Please refer to the KAPA Single-Indexed Adapter Technical Data Sheet (include link) for information about barcode sequences, pooling, kit configurations, formulation, and dilution of KAPA Single-Indexed Adapters.

What QC testing is performed on KAPA Adapters?

KAPA Adapters undergo extensive qPCR- and sequencing-based functional and QC testing to confirm:

  • optimal library construction efficiency
  •  minimal levels of adapter-dimer formation
  • nominal levels of barcode cross-contamination

Library construction efficiency and adapter-dimer formatin are assessed in a low-input library construction workflow. The conversion rate achieved in the assay indicates library construction efficiency. This is calculated by measuring the yield of adapter-ligated library (before any amplification) by qPCR (using the KAPA Library Quantification Kit), and expressing this as a % of input DNA. To assess adapter-dimer formation, a modified library construction protocol— designed to measure adapter dimer with high sensitivity—is used. Pass criteria for this assay translate to adapter-dimer carry-over in a standard workflow in the range of 0 – 2 %.

Barcode cross-contamination is assessed by sequencing. Each adapter is ligated to a unique, synthetic insert of known sequence, using a standard library construction protocol. These constructs pooled and sequenced on a MiSeq. For every barcode, the number of reads (in the range of 115,000 – 500,000) associated with each insert is counted, and the total % correct inserts calculated. Contamination of any barcode with any other single barcode is guaranteed to be <0.25%. The total level of contamination for any barcode is typically in the range of 0.1 – 0.5%. This assay is unable to distinguish between chemical cross-contamination and adapter “cross-talk”, and measures the total number of incorrect inserts resulting from both phenomena.

What is the fragmentation protocol with this kit?

RNA is fragmented using high temperature in the presence of magnesium. Depending on the origin and integrity of the input RNA, and the intended application, different RNA fragmentation protocols are provided to obtain the required insert size distribution. For intact RNA such as that extracted from fresh/frozen tissue, longer fragmentation is required at higher temperatures. For degraded or fragmented RNA (e.g. from older samples or formalin-fixed-paraffin-embedded (FFPE) tissue), use a lower temperature and/or shorter times. The table below outlines various fragmentation parameters depending on the input RNA and the desired insert size.

Input RNA Desired Insert Size Fragmentation and Priming
Intact 100-200 bp 8 min @ 94˚C
200-300 bp 6 min @ 94˚C
300-400 bp 6 min @ 85˚C
Partially degraded 100-300 bp 1-6 min @ 85˚C
Degraded * 100-200 bp 30 sec @ 65˚C

* This facilitates annealing of the random primers, and will not result in any significant additional fragmentation of the RNA.

How should I measure the final library?

The size distribution of the double-stranded cDNA and/or final amplified library should be confirmed with an electrophoretic method. The quantification of the library should be done with a qPCR based quantification kit such as the KAPA Library Quantification Kits for Illumina platforms. These kits employ primers based on the Illumina flow cell oligos, and can be used to quantify libraries that are ready for flow-cell amplification.

Which polymerase is used for amplification?

KAPA HiFi HotStart is the enzyme provided in the KAPA HiFi HotStart ReadyMix. This is a novel B-family DNA polymerase engineered for low-bias, high fidelity PCR and is the reagent of choice for NGS library amplification1,2,3.

  1. Oyola, S.O. et al. BMC Genomics 13, 1 (2012).
  2. Quail M.A. et al. Nature Methods 9, 10-11 (2012).
  3. Quail M.A. et al. BMC Genomics 13, 341 (2012).

How many cycles should I use when amplifying my adapter-ligated library?

To minimize over-amplification and associated unwanted artifacts, the number of PCR cycles should be optimized to produce a final amplified library with a concentration range of 10-30 ng/µL which is equivalent to 0.5-1.5 µg of DNA per 50 µL reaction. The number of cycles recommended below should be used as a guide for library amplification, but cycle numbers may have to be adjusted (± 4 cycles) depending on library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

Input RNA Number of Cycles
10-50 ng 14
50-200 ng 12
200-400 ng 10

What are the storage conditions for this kit?

The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and storage. Upon receipt, immediately store enzymes and reaction buffer components at -20°C in a constant-temperature freezer. The PEG/NaCl Solution may be stored at 4°C for up to 2 months. When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.

Ordering

Kits include reagents for RNA fragmentation, 1st strand cDNA synthesis and 2nd strand synthesis/marking, and cDNA library preparation, including A-tailing, ligation and library amplification. Kits with reagents for mRNA capture are also available.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8400
07962142001
Stranded RNA-Seq Kit
24 libraries
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KK8401
07962169001
Stranded RNA-Seq Kit
96 libraries
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KK8420
07962193001
KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads
24 libraries
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KK8421
07962207001
KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads
96 libraries
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Accessory Products

KAPA Single-Indexed Adapter Set A contains indices 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18 and 19, whereas Set B contains indices 1, 3, 8, 9, 10, 11, 20, 21, 22, 23, 25, 27. All KAPA Single- and Dual-Indexed Adapter Kits contain KAPA Adapter Dilution Buffer. KAPA Dual-Indexed Adapter Kits also contain three additional sealing films to support multiple use.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8000
07983271001
KAPA Pure Beads (5 mL)
5 mL
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KK8001
07983280001
KAPA Pure Beads (30 mL)
30 mL
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KK8002
07983298001
KAPA Pure Beads (60 mL)
60 mL
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KK8722
08278555702
KAPA Dual-Indexed Adapter Kit, (15 µM)
96 adapters x 20 µl each
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KK8721
08278539001
KAPA Adapter Dilution Buffer (25 mL)
25 mL
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for pricing