Product Description
The KAPA mRNA HyperPrep Kit for Illumina sequencing contains all of the buffers and enzymes required for poly(A) mRNA capture and the rapid construction of stranded mRNA-Seq libraries from 50 ng – 1 µg of intact total RNA via the following steps:
- mRNA capture using magnetic oligo-dT beads;
- fragmentation using heat and magnesium;
- 1st strand cDNA synthesis using random priming;
- combined 2nd strand synthesis and A-tailing, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporates dUTP into the second cDNA strand, and adds dAMP to the 3' ends of the resulting dscDNA;
- adapter ligation, where dsDNA adapters with 3' dTMP overhangs are ligated to library insert fragments; and
- 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, allowing strand-specific sequencing.
The kit provides KAPA Pure Beads for reaction cleanups, along with all of the enzymes and buffers required for mRNA capture, cDNA synthesis, library construction and amplification, but does not include RNA or adapters. KAPA Adapters are sold separately.
Reaction buffers are supplied in convenient formats comprising all of the required reaction components. This minimizes the risk of RNase contamination, ensures consistent and homogenous reaction composition, and improves uniformity among replicate samples. Similarly, a single enzyme mixture is provided for each step of the library construction process, reducing the number of pipetting steps.
In order to maximize sequence coverage uniformity and to maintain relative transcript abundance, it is critical that library amplification bias be kept to a minimum. KAPA HiFi DNA Polymerase has been designed for low-bias, high- fidelity PCR, and is the polymerase of choice for NGS library amplification1, 2, 3, 4. The KAPA mRNA HyperPrep Kit includes KAPA HiFi HotStart ReadyMix (2X) and Library Amplification Primer Mix (10X) for library amplification.
- Oyola, S.O., et al., BMC Genomics 13, 1 (2012).
- Quail, M.A., et al., Nature Methods 9, 10-11 (2012).
- Quail, M.A., et al., BMC Genomics 13, 341 (2012).
- Ross, M.G., et al., Genome Biology 14, R51 (2013).
Product Applications
The KAPA mRNA HyperPrep Kit is designed for both manual and automated NGS library construction from 50 ng-1 µg of intact total RNA. The protocol is applicable to a wide range of RNA-Seq applications, including:
•gene expression;
•single nucleotide variation (SNV) discovery;
•splice junction and gene fusion identification; and
•characterization of polyadenylated RNAs.
This kit is not compatible with small RNAs < 100 bp in length.
Process Workflow
Library Construction Protocol
- Reagent Preparation
This protocol takes approximately 5.5 h to complete. Ideally, master mixes for the various steps in the process should be prepared as required.
For maximum stability and shelf-life, the enzymes and reaction buffers are supplied separately in the KAPA mRNA HyperPrep Kit.
For a streamlined protocol, a reagent master mix with a minimum of 10% excess is prepared for each of these enzymatic steps, as outlined in Tables 2-5. Volumes of additional reagents required for the KAPA mRNA HyperPrep Kit protocol are listed in Table 6.
Always ensure that KAPA Pure Beads and PEG/NaCl Solution are fully equilibrated to room temperature before use.
Table 2. 1st strand synthesis
Table 3. 2nd strand synthesis and A-tailing
Table 4. Adapter ligation
Table 5. Library amplification
Table 6. Volumes of additional reagents required
- mRNA Capture
This protocol requires 50 ng-1 µg of intact total RNA, in 50 µL of RNase-free water. Degraded or fragmented total RNA will result in significant 3'-bias.
This protocol has been optimized to isolate mature mRNA from total RNA through two subsequent capture steps using mRNA Capture Beads. Other RNA molecules with homopolymeric adenosine regions may also be isolated.
RNA samples should only be kept on ice where specified in this protocol, since low temperatures may promote non-specific capture, resulting in increased rRNA levels in the captured mRNA.
Before starting, equilibrate mRNA Capture Beads, mRNA Bead Binding Buffer, mRNA Bead Wash Buffer and Fragment, Prime and Elute Buffer (2X) to room temperature.
Before use, beads must be washed with mRNA Bead Binding Buffer (Steps 2.1-2.5).
- mRNA Elution, Fragmentation and Priming
- 1st Strand Synthesis
- 2nd Strand Synthesis and A-tailing
- Adapter Ligation
- 1st Post-ligation Cleanup
- 2nd Post-ligation Cleanup
- Library Amplification
- Library Amplification Cleanup