The streamlined, one-tube KAPA Hyper Prep protocol offers fast Library Prep turnaround time, with minimal hands-on time. Offering complete library construction in less than 3 hours (library amplification may be omitted for PCR-free workflows) and bead-based size selection steps that can be incorporated to achieve the appropriate final library fragment-size distribution.
The KAPA RNA HyperPrep Kit for Illumina sequencing contains all of the buffers and enzymes required for the rapid construction of stranded RNA-Seq libraries from 1 – 100 ng of purified total, rRNA-depleted, or poly(A)- enriched RNA via the following steps:
- 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 for stranded RNA sequencing, 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 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, highfidelity PCR, and is the polymerase of choice for NGS library amplification[1,2,3,4]. The KAPA RNA HyperPrep Kit includes KAPA HiFi HotStart ReadyMix (2X) and Library Amplification Primer Mix (10X) for library amplification.
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).