KAPA Stranded RNA-Seq Kits

Overview

The KAPA Stranded RNA-Seq Kit combines the use of a “with bead” protocol with KAPA HiFi DNA Polymerase selected through our directed evolution technology for high efficiency, industry-leading fidelity and low-bias amplification to provide superior quality RNA libraries with flexible input amounts. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads, available separately.

Benefits of KAPA Stranded RNA-Seq Kits

  • Tunable fragmentation for various sequencing applications
  • High yield of adapter-ligated library
  • High coverage of GC-rich and low-abundance transcripts*
  • Qualified automation methods

 

Product Highlights

High coverage uniformity

  • Comparable transcript coverage at various inputs
  • Reproducible and uniform distribution of reads across the transcript

High isoform discovery and sensitivity with target capture

  • Increased isoform detection and sensitivity
  • Selection of biologically-relevant transcripts of interest

*Data on file.

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.

Specifications
Spec
Description
Compatible Platform
Illumina HiSeq,NextSeq,MiSeq and GAIIX
Starting Material
Total RNA,mRNA, or ribosomal depleted RNA
Input Amount 10ng-400ng

Components

Components

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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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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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KK8710
08005770001
KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM)
24 adapters x 40 µl each Contact Us
KK8711
08005788001
KAPA Single-Indexed Adapter Kit, Set A (1.5 µM)
12 adapters x 40 µl each
Contact Us
KK8712
08005796001
KAPA Single-Indexed Adapter Kit, Set B (1.5 µM)
12 adapters x 40 µl each
Contact Us
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 Contact Us
  • Gene expression
  • Polymorphism detection
  • Genome annotation
  • Alternative splicing
  • RNA editing

No, these kits are not compatible with small RNA.

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

  • 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.

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.

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.

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.

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.

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 TruSeq,  SeqCap EZ, Agilent SureSelect, and other similar library construction and target capture workflows. 

 

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.

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.

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.

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).

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

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.