Product Description
The KAPA Stranded RNA-Seq Library Preparation Kit contains all of the buffers and enzymes required for the construction of libraries from 10 – 400 ng of total, rRNAdepleted, or poly(A)-enriched RNA via the following steps:
- 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 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; 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 all of the enzymes and buffers required for cDNA synthesis and library construction but does not include RNA, adapters, or beads. KAPA Pure Beads 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 has been designed for low-bias, highfidelity PCR, and is the polymerase of choice for NGS library amplification1,2,3,4. KAPA Stranded RNA-Seq Library Preparation Kits include 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
This KAPA Stranded RNA-Seq Library Preparation Kit is designed for both manual and automated NGS library construction from 10 – 400 ng of total, rRNA-depleted, or poly(A)-enriched RNA. The protocol is applicable to a wide range of RNA-seq applications, including:
• targeted RNA-seq
• whole transcriptome
• gene expression analysis of high- and low-quality RNA samples (e.g., extracted from FFPE tissue)
• single nucleotide variation (SNV) discovery
• splice junction and gene fusion identification.
Note: The KAPA Stranded RNA-Seq Library Preparation Kit is not compatible with small RNAs <100 bp in length.
Process Workflow
Library Construction Protocol
- Reagent Preparation
This protocol takes 6 – 8 hrs to complete. Ideally,master mixes for the various steps in the process should be prepared as required.
For maximum stability and shelf-life, enzymes and\reaction buffers are supplied separately in KAPAStranded RNA-Seq Library Preparation Kit. For a streamlined “with-bead” protocol, a reagent master mix is prepared for each of these enzymatic steps, as outlined in Tables 2 – 6. Volumes of additional reagents required for the KAPA Stranded RNA-Seq Library Preparation Kit protocol are listed in Table 7.
In some cases, master mixes may be constituted with varying proportions of the total final water requirement. In the examples given in the tables below, all the required water is included in each master mix, allowing the entire reaction mix to be added in a single pipetting step.
At the safe stopping point at A-tailing, a portion of the water and reaction buffer are added to the beads for storage at 2°C to 8°C for ≤24 hrs.To resume library construction, prepare the master mix with the remaining volume of water and reaction buffer, and the required volume of enzyme. Recommendations on how to formulate the master mix after this safe stopping point are provided in Table 4B.
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 marking
Table 4A. A-tailing (uninterrupted protocol)
Table 4B. A-tailing (safe stopping point)
Table 5. Adapter ligation
Table 6. Library amplification
Table 7. Volumes of additional reagents required
- RNA Fragmentation
This protocol requires 10 – 400 ng of total, rRNAdepleted,or poly(A)-enriched RNA, in 10 μL of RNase-free water.
Input RNA is suspended in Fragment, Prime and Elute Buffer (1X) and fragmented to the desired size by incubation at high temperature.
- 1st Strand Synthesis
- 2nd Strand Synthesis and Marking
- 2nd Strand Synthesis and Marking Cleanup
- A-Tailing
A-tailing is performed either directly after the 2nd Strand Synthesis and Marking Cleanup
(step 5), or after the Safe Stopping Point, where beads were resuspended in A-Tailing Buffer (1X) and stored at 2°C to 8°C for ≤24 hrs. Depending on your chosen workflow, proceed with either A-Tailing Immediately (step 6A) or A-tailing after Safe Stopping Point (step 6B).
6A. A-tailing Immediately
6B. A-tailing after Safe Stopping Point
- Adapter Ligation
- 1st Post-ligation Cleanup
- 2nd Post-ligation Cleanup
- Library Amplification
- Library Amplification Cleanup