KAPA Stranded RNA-Seq Kits
with RiboErase (HMR)

Overview

The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) targets and depletes rRNA from various sample types (human, mouse, rat) and provides more consistent and effective rRNA depletion than traditional bead-based capture methods.* Efficient rRNA depletion prior to library preparation results in increased coverage of transcripts of interest, including noncoding and precursor transcripts. This kit is suitable for total RNA input from human, mouse and rat species only. 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 with RiboErase

  • Up to 99.98% rRNA depletion from various sample types*
  • Compatible with degraded inputs, including FFPE
  • High coverage of GC-rich and low-abundance transcripts
  • Qualified automation methods

Featured Webinar

KAPA RNA-Seq with RiboErase: Providing a more comprehensive view of the transcriptome

Presenter: Jennifer Pavlica, Roche

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* Data on file.

Kits can be stored for up to 10 months at -20ºC.

Kits contain KAPA Hybridization Buffer, KAPA Hybridization Oligos (Human/Mouse/Rat), KAPA Depletion Buffer, KAPA RNase H, KAPA DNase Buffer, KAPA Dnase, KAPA Fragment, Prime and Elute Buffer (2X), KAPA 1st Strand Synthesis Buffer, KAPA Script, KAPA 2nd Strand Marking Buffer, KAPA 2nd Strand Synthesis Enzyme Mix, KAPA A-Tailing Buffer (10X), KAPA A-Tailing Enzyme, KAPA Ligation Buffer (5X), KAPA DNA Ligase, KAPA PEG/NaCl SPRI Solution, KAPA Library Amplification Primer Mix (10X) and KAPA HiFiHotStartReadyMix (2X).

Specifications
Spec
Description
Compatible Platform
Illumina HiSeq, NextSeq and MiSeq
Library Type
RNA
Starting Material
High-quality Total RNA
Input Amount
100 ng - 1 µg

Components

Components


Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK8483
07962282001
KAPA Stranded RNA-Seq Kit with RiboErase (HMR)
24 reactions
Login for pricing
KK8484
07962304001
KAPA Stranded RNA-Seq Kit with RiboErase (HMR)
96 reactions
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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 Login for pricing
KK8001
07962304001
KAPA Stranded RNA-Seq Kit with RiboErase (HMR)
30 mL
Login for pricing
KK8002
07983298001
KAPA Pure Beads (60 mL)
60 mL Login for pricing
KK8710
08005770001
KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM)
24 adapters x 40 µl each Login for pricing
KK8711
08005788001
KAPA Single-Indexed Adapter Kit, Set A (1.5 µM)
12 adapters x 40 µl each
Login for pricing
KK8712
08005796001
KAPA Single-Indexed Adapter Kit, Set B (1.5 µM)
12 adapters x 40 µl each
Login for pricing
KK8722
08278555702
KAPA Dual-Indexed Adapter Kit, (15 µM)
96 adapters x 20 µl each
Login for pricing
KK8721
08278539001
KAPA Adapter Dilution Buffer (25 mL)
25 mL Login for pricing
  • Gene expression
  • Polymorphism detection
  • Genome annotation
  • Alternative splicing
  • RNA editing

No, these kits are not compatible with small RNA.

100 – 1000 ng of purified total RNA in ≤10 µL of water.

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.

While the poly(A) capture method used in an mRNA-Seq workflow is useful when specifically interrogating mRNA species, the workflow does bias towards exonic transcripts.  If you wish to obtain a more accurate representation of the whole transcriptome, with only the rRNA sequences removed, then ribodepletion is the better option.  It allows for more accurate representation of intronic and intergenic regions, which is where many long non-coding transcripts are found.
 

Additionally, the poly(A) capture approach makes it suboptimal for use degraded RNA, where there is the possibility of strand breaks between the 3’ polyadenylation and the rest of the transcript.

Unfortunately, we do not offer a globin depletion solution at this time.

The DNA oligos used for depletion were specifically designed with human, mouse, and rat rRNA sequences in mind, and the kit has only been validated for these species.

While the DNA oligos used for depletion were specifically designed with human, mouse, and rat rRNA sequences in mind, there is a potential for the kit to be used with other species where there is a good amount of homology in the rRNA sequences in comparison to human, mouse, or rat.  Please contact kapabiosystems.com/support with specific species inquiries.

Yes, however library quality is linked to both input RNA quality and quantity.

Step 1: Ribosomal RNA (rRNA) depletion through:

  • Hybridization of DNA oligonucleotides complementary to the ribosomal regions.
  • Enzymatic depletion of rRNA with the RNase H.
  • Removal of DNA oligonucleotides with a DNase I digestion.

Step 2: RNA fragmentation using heat and magnesium.

Step 3: cDNA Synthesis:

  • 1st Strand Synthesis using random priming.
  • 2nd Strand Synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA) and incorporates dUTP in the second cDNA strand.

Step 4: A-tailing to add dAMP to the 3′-ends of the dscDNA library fragments.

Step 5: Adapter ligation, where dsDNA adapters with 3′-dTMP overhangs are ligated to A-tailed library insert fragments.

Step 6: 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.

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 rRNA depletion through library amplification can be performed in approximately 10 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 with RiboErase. However, kits are also compatible with non-indexed, single-indexed, and dual-indexed adapters that are routinely used in Illumina TruSeq, 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.

 

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 rRNA depletion reaction.

 

Quantity of starting material Adapter stock concentration Adapter Concentration in ligation reaction
501-1000 ng 280 nM 20 nM
251-500 ng 210 nM 15 nM
100-250 ng 140 nM 10 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 for information about barcode sequences, pooling, kit configurations, formulation, and dilution of KAPA Single-Indexed 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.

RNA is fragmented using a 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 desired 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 bp200-300 bp 8 min @ 94˚C6 min @ 94˚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).
  4. Ross, M.G., et al., Genome Biology 14, R51 (2013).

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 depending on library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

 

Input RNA Number of Cycles
100-250 ng 12-16
251-500 ng 10-13
501-1000 ng 8-11

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.