In this protocol

适用说明:本说明书适用于KAPA Stranded mRNA-Seq试剂盒 (07962193001和07962207001) 和KAPA mRNA Capture试剂盒 (07962231001和07962240001).
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Product Description

The KAPA Stranded mRNA-Seq Kit contains all of the buffers and enzymes required for poly(A) mRNA capture and construction of stranded mRNA-seq libraries from 100 ng – 4 μg of intact, total RNA via the following steps:

  1. mRNA capture using magnetic oligo-dT beads;
  2. fragmentation using heat and magnesium;
  3. 1st strand cDNA synthesis using random priming;
  4. 2nd strand synthesis and marking, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA),and incorporates dUTP into the 2nd cDNA strand;
  5. A-tailing, to add dAMP to the 3'-ends of the dscDNA library fragments;
  6. adapter ligation, where dsDNA adapters with 3'-dTMP overhangs are ligated to A-tailed library insert fragments; and
  7. 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.

This kit provides all of the enzymes and buffers required for mRNA enrichment, cDNA synthesis, and library construction and amplification, 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 is designed for low-bias, highfidelity PCR, and is the polymerase of choice for NGS library amplification1,2,3,4. KAPA Stranded mRNA-Seq 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 (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

The KAPA Stranded mRNA-Seq Kit is designed for both manual and automated NGS library construction from 100 ng – 4 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
• characterization of polyadenylated RNAs.

Process Workflow


Library Construction Protocol

  1. Reagent Preparation
    This protocol takes 8 – 10 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 the KAPA Stranded mRNA-Seq Kit. For a streamlined“with-bead” protocol, a reagent master mix with a minimum of 10% excess is prepared for each of these enzymatic steps, as outlined in Tables 2 – 6.

    Volumes of additional reagents required for the KAPA Stranded mRNA-Seq 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 the 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. mRNA Capture
    This protocol requires 100 ng – 4 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 the 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 in the captured mRNA.

    Before starting, equilibrate mRNA Capture Beads, mRNA Bead Binding Buffer, mRNA Bead Wash Buffer and Fragment, Prime and Elute Buffer to room temperature.

    Before use, beads must be washed with mRNA Bead Binding Buffer (steps 2.1 – 2.5).
    2.1
    Resuspend the mRNA Capture Beads thoroughly by pipetting up and down gently, or by using a vortex mixer on a low to medium speed setting to prevent foaming. High-speed vortexing or shaking should be avoided to prevent foaming. Refer to Important Parameters: mRNA Capture Beads (p. 3) for more information.
    2.2
    For each library to be prepared, transfer 52.5 μL (50 μL + 5% excess) of the resuspended mRNA Capture Beads into an appropriate tube. When preparing multiple libraries, beads for up to 24 libraries (1,260 μL) may be washed in a single tube. When preparing more than 24 libraries, wash beads in multiple batches. Please refer to Important Parameters: mRNA Capture Beads (p. 3) for additional recommendations regarding bulk bead washing.
    2.3
    Place the tube on a magnet and incubate at room temperature until the solution is clear. Carefully remove and discard the supernatant, and replace it with an equal volume of mRNA Bead Binding Buffer (52.5 μL per library).
    2.4
    Remove the tube from the magnet and resuspend the beads by pipetting up and down, or by low to medium speed vortexing. Be careful to avoid producing excessive foam.
    2.5
    Place the tube on the magnet and incubate at room temperature until the solution is clear. Carefully remove and discard the supernatant, and replace it with an equal volume of mRNA Bead Binding Buffer (52.5 μL per library).
    2.6
    Remove the tube from the magnet and resuspend the beads by pipetting up and down, or by low to medium speed vortexing. Be careful to avoid producing excessive foam.
    2.7
    For each RNA sample to be captured, transfer 50 μL of resuspended mRNA Capture Beads into individual tubes or wells of a plate.
    2.8
    To each well/tube, add 50 μL of the appropriate RNA sample (in RNase-free water).
    2.9
    Mix thoroughly by gently pipetting up and down several times.
    2.10
    Place the plate/tube(s) in a thermocycler and perform the 1st mRNA capture as follows:


    2.11
    Place the plate/tube(s) containing the mixture of mRNA Capture Beads and RNA on a magnet and incubate at room temperature until the solution is clear. Remove and discard the supernatant.
    2.12
    Remove the plate/tube(s) from the magnet and resuspend thoroughly in 200 μL of mRNA Bead Wash Buffer by pipetting up and down several times.
    2.13
    Place the plate/tube(s) on the magnet and incubate at room temperature until the solution is clear.Remove and discard the supernatant.
    2.14
    Resuspend the beads in 50 μL of RNase-free water.
    2.15
    Place the plate/tube(s) in a thermocycler and perform the 2nd mRNA capture as follows:


    2.16
    Add 50 μL of Bead Binding Buffer to the mixture of mRNA Capture Beads and RNA and mix thoroughly by gently pipetting up and down several times.
    2.17
    Incubate the plate/tube(s) at 20°C for 5 min.
    2.18
    Place the plate/tube(s) on the magnet and incubate at room temperature until the solution is clear.Remove and discard the supernatant.
    2.19
    Remove the beads from the magnet and resuspend in 200 μL of mRNA Bead Wash Buffer by pipetting up and down several times.
    2.20
    Place the plate/tube(s) on the magnet andincubate at room temperature until the solution is clear. Remove and discard the entire volume of supernatant.
    Caution: carryover of mRNA Bead Wash Buffer may inhibit 1st Strand CDNA Synthesis.

  3. mRNA Elution, Fragmentation and Priming
    3.1
    Prepare the required volume of Fragment, Prime and Elute Buffer (1X) at room temperature as follows:


    3.2
    Thoroughly resuspend the mRNA Capture Beads with captured mRNA prepared in step 2.20 above in 22 μL of Fragment, Prime and Elute Buffer (1X).

    SAFE STOPPING POINT
    Resuspended beads with captured mRNA may be stored at 2 °C to 8 °C for ≤ 24 hrs. Do not freeze the samples as this will damage the beads. When ready, proceed to step 3.3 below.

    3.3
    Place the plate/tube(s) in a thermocycler and carry out the fragmentation and priming program given in the table below:


    3.4
    Immediately place the plate/tube(s) on a magnet tocapture the beads, and incubate until the liquid is clear.
    Caution: to prevent hybridization of poly(A)-rich RNA to the capture beads, do not allow the sample to cool before placing on the magnet.
    3.5
    Carefully remove 20 μL of the supernatantcontaining the eluted, fragmented, and primed RNA into a separate plate or tube.
    3.6
    Place the plate/tube(s) on ice and proceed immediately to 1st Strand Synthesis (step 4).

  4. 1st Strand Synthesis
    4.1
    On ice, assemble the first strand synthesis reaction as follows:


    4.2
    Keeping the plate/tube(s) on ice, 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
    Place the plate/tube(s) on ice and proceed immediately to 2nd Strand Synthesis and Marking (step 5).

  5. 2nd Strand Synthesis and Marking
    5.1
    On ice, assemble the second strand synthesis and marking reaction as follows:


    5.2
    Keeping the plate/tube(s) on ice, mix thoroughly by gently pipetting the reaction up and down several times.
    5.3
    Incubate the plate/tube(s) using the following protocol:


    5.4
    Proceed immediately to 2nd Strand Synthesis and Marking Cleanup (step 6).

  6. 2nd Strand Synthesis and Marking Cleanup
    6.1
    Perform a 1.8X bead-based cleanup by combining the following:


    6.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    6.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    6.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    6.5
    Carefully remove and discard 160 μL of supernatant.
    6.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    6.7
    Incubate the plate/tube(s) at room temperature for ≥30 sec.
    6.8
    Carefully remove and discard the ethanol.
    6.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    6.10
    Incubate the plate/tube(s) at room temperature for ≥30 sec.
    6.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    6.12
    Dry the beads at room temperature until all of the ethanol has evaporated.
    Caution: over-drying the beads may result in reduced yield.
    6.13
    Proceed immediately to A-tailing Immediately (step 7A), or follow the safe stopping point instructions.
      
    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 7B).

  7. A-tailing
    A-tailing is performed either directly after the 2nd Strand Synthesis and Marking Cleanup, 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 7A) or A-tailing after Safe Stopping Point (step 7B).

    7A. A-tailing Immediately
    7A.1
    Assemble the A-tailing reaction as follows:


    7A.2
    Mix thoroughly by pipetting up and down several times.
    7A.3
    Incubate the plate/tube(s) using the following protocol:


    7A.4
    Proceed immediately to Adapter Ligation (step 8).

    7B. A-tailing after Safe Stopping Point
    7B.1
    To resume library preparation, combine the following reagents to perform A-tailing:


    7B.2
    Mix thoroughly by pipetting up and down several times.
    7B.3
    Incubate the plate/tube(s) using the following protocol:


    7B.4
    Proceed immediately to Adapter Ligation (step 8).

  8. Adapter Ligation
    8.1
    Dilute adapters in preparation for ligation targeting the following concentrations:


    8.2
    Set up the adapter ligation reactions as follows:


    8.3
    Mix thoroughly by pipetting up and down several times to resuspend the beads.
    8.4
    Incubate the plate/tube(s) at 20°C for 15 min.
    8.5
    Proceed immediately to 1st Post-Ligation Cleanup (step 9).

  9. 1st 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 5 – 15 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 135 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 50 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.

    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 10).

  10. 2nd Post-ligation Cleanup
    10.1
    Perform a 1X bead-based cleanup by combining the following:


    10.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    10.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    10.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    10.5
    Carefully remove and discard 95 μL of supernatant.
    10.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    10.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    10.8
    Carefully remove and discard the ethanol.
    10.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    10.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    10.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    10.12
    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.
    10.13
    Remove the plate/tube(s) from the magnet.
    10.14
    Thoroughly resuspend the beads in 22 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    10.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    10.16  Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    10.17
    Transfer 20 μL of the clear supernatant to a new%2
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