The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) contains all of the buffers and enzymes required for depletion of ribosomal RNA (rRNA) followed by construction of stranded RNA-seq libraries from 100 ng – 1 µg of total RNA via the following steps:
- depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides, respectively;
- fragmentation using heat and magnesium;
- 1st strand cDNA synthesis using random priming;
- 2nd strand synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the 2nd 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; and
- library amplification, to amplify library fragments carrying appropriate adapter sequences at both ends using highfidelity, low-bias PCR. The strand marked with dUTP is not amplified, allowing strand-specific sequencing.
The kit provides all of the enzymes and buffers required for rRNA depletion, cDNA synthesis, and library construction and amplification, but does not include RNA, adapters, or beads. KAPA Pure Beads (KK8000, KK8001, KK8002) and 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 is designed for low-bias, high-fidelity PCR, and is the polymerase of choice for NGS library amplification.[1,2,3,4] The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) 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).