In this protocol

适用说明:本说明书适用于KAPA Stranded RNA-Seq Library Preparation Kit Illumina® Platforms (KK8400,kk8401)。
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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:

  1.  fragmentation using heat and magnesium;
  2. 1st strand cDNA synthesis using random priming;
  3. 2nd strand synthesis and marking, which converts the cDNA:RNA hybrid to double- stranded cDNA (dscDNA),and incorporates dUTP into the second cDNA strand;
  4.  A-tailing, to add dAMP to the 3'-ends of the dscDNA library fragments;
  5. adapter ligation, where dsDNA adapters with 3'-dTMP overhangs are ligated to A-tailed library insert fragments; and
  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, 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. 

  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).

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

  1.  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


  2. 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.
    2.1
    repare the required volume of Fragment, Prime and Elute Buffer (1X) at room temperature as follows:


    2.2
    Mix thoroughly by gently pipetting the reaction up and down several times.
    2.3
    Place the plate/tube(s) in a thermocycler and carry out the fragmentation and priming program as follows:


    2.4
    Place the plate/tube(s) on ice and proceed immediately to 1st Strand Synthesis (step 3).
  3. 1st Strand Synthesis
    3.1
    On ice, assemble the first strand synthesis reaction as follows:

    3.2
    Keeping the plate/tube(s) on ice, mix thoroughly by gently pipetting the reaction up and down several times.
    3.3
    Incubate the plate/tube(s) using the following protocol:

    3.4
    Place the plate/tube(s) on ice and proceed immediately to 2nd Strand Synthesis and Marking (step 4).

  4. 2nd Strand Synthesis and Marking
    4.1
    Assemble the second strand synthesis and marking reaction as follows:


    4.2
    Mix thoroughly by gently pipetting the reaction up and down several times.
    4.3
    Incubate the plate/tube(s) using the following protocol:


    4.4
    Proceed immediately to 2nd Strand Synthesis and Marking Cleanup (step 5).

  5.  2nd Strand Synthesis and Marking Cleanup
    5.1
    Perform a 1.8X bead-based cleanup by combining the following:


    5.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    5.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    5.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    5.5
    Carefully remove and discard 160 μL of supernatant.
    5.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.8
    Carefully remove and discard the ethanol.
    5.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    5.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    5.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    5.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
    5.13
    Proceed immediately to A-tailing Immediately (step 6A), or follow the Safe Stopping Point instructions below.

    SAFE STOPPING POINT
    Resuspend the beads in 15 μL A-Tailing Buffer (1X) (Table 4B), cover the reaction and store at 2°C to 8°C for ≤24 hrs. Do not freeze the samples as this will damage the KAPA Pure Beads. When ready, proceed to A-tailing after Safe Stopping Point (step 6B).

  6. 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
    6A.1
    Assemble the A-tailing reaction as follows:


    6A.2
    Mix thoroughly by pipetting up and down several times.
    6A.3
    Incubate the plate/tube(s) using the following protocol:


    6A.4
    Proceed immediately to Adapter Ligation (step 7).

    6B. A-tailing after Safe Stopping Point
    6B.1
    To resume library preparation, combine thefollowing reagents to perform A-tailing:


    6B.2
    Mix thoroughly by pipetting up and down several times.
    6B.3
    Incubate the plate/tube(s) using the following protocol:


    6B.4
    Proceed immediately to Adapter Ligation (step 7).

  7.  Adapter Ligation
    7.1
    Dilute adapters in preparation for ligation, targeting the following concentrations:


    7.2
    Set up the adapter ligation reactions as follows:


    7.3
    Mix thoroughly by pipetting up and down several times to resuspend the beads.
    7.4
    Incubate the plate/tube(s) at 20 °C for 15 min.
    7.5
    Proceed immediately to 1st Post-ligation Cleanup (step 8).

  8. 1st Post-ligation Cleanup
    8.1
    Perform a 1X bead-based cleanup by combining the following:


    8.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    8.3
    Incubate the plate/tube(s) at room temperature for 5– 15 min to bind DNA to the beads.
    8.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    8.5
    Carefully remove and discard 135 μL of supernatant.
    8.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    8.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    8.8
    Carefully remove and discard the ethanol.
    8.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    8.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec. 
    8.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    8.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    8.13
    Remove the plate/tube(s) from the magnet.
    8.14
    Thoroughly resuspend the beads in 50 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    8.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.

    SAFE STOPPING POINT

    The solution with resuspended beads can be stored at 2°C to 8°C for ≤24 hrs. Do not freeze the beads, as this can result in dramatic loss of DNA. When ready, proceed to 2nd Post-ligation Cleanup (step 9).

  9. 2nd Post-ligation Cleanup
    9.1
    Perform a 1X bead-based cleanup by combining the following:


    9.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    9.3
    Incubate the plate/tube(s) at room temperature for 2 min to bind DNA to the beads.
    9.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    9.5
    Carefully remove and discard 95 μL of supernatant.
    9.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    9.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    9.8
    Carefully remove and discard the ethanol.
    9.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    9.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    9.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    9.12
    Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    9.13
    Remove the plate/tube(s) from the magnet.
    9.14
    Thoroughly resuspend the beads in 22 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    9.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    9.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    9.17
    Transfer 20 μL of the clear supernatant to a new plate/tube(s) and proceed to Library Amplification (step 10).

    SAFE STOPPING POINT
    The purified, adapter-ligated library DNA may be stored at 2°C to 8°C for ≤1 week, or frozen at -15°C to -25°C for ≤1 month. When ready, proceed to Library Amplification (step 10).

  10. Library Amplification
    10.1
    Assemble each library amplification reaction as follows:


    10.2
    Mix well by pipetting up and down several times.
    10.3
    Amplify the library using the following thermocycling profile:


    10.4
    Proceed immediately to Library Amplification Cleanup (step 11).

  11. Library Amplification Cleanup
    11.1
    Perform a 1X bead-based cleanup by combining the following:


    11.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    11.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    11.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    11.5
    Carefully remove and discard 95 μL of supernatant.
    11.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    11.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    11.8
    Carefully remove and discard the ethanol.
    11.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    11.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    11.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    11.12
    Dry the beads at room temperature for 3 – 5 min,or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
    11.13
     Remove the plate/tube(s) from the magnet.
    11.14
    Thoroughly resuspend the dried beads in 22 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    11.15
     Incubate the plate/tube(s) at room temperature for2 min to elute DNA off the beads.
    11.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    11.17
    Transfer 20 μL of the clear supernatant to a new plate/tube(s) and store the purified, amplified libraries at 2°C to 8°C for ≤ 1 week, or at -15°C to -25°C.
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