In this protocol

产品说明:本说明书适用于KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin Illumina® Platforms试剂盒(货号为KK8485、KK8400、 KK8481、 KK8478、KK8486、KK8401、KK8482 和KK8479)。
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Product Description

The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin for Illumina sequencing contains all of the buffers and enzymes required for depletion of rRNA and globin mRNA transcripts and the construction of stranded RNASeq libraries from 100 ng – 1 μg of purified, blood-derived total RNA via the following steps:

  1. depletion of rRNA and globin mRNA by hybridization of complementary DNA oligonucleotides and RNase H digestion to remove RNA duplexed to DNA, followed by DNase treatment to remove DNA oligonucleotides;
  2. random 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 second 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.

The kit provides all of the enzymes and buffers required for rRNA and globin mRNA depletion, cDNA synthesis, library construction and amplification. 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) Globin 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).


Product Applications

The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin is designed for both manual and automated NGS library construction from 100 ng – 1 μg of blood-derived total RNA.

The kit depletes both cytoplasmic (5S, 5.8S, 18S, and 28S), and mitochondrial (12S and 16S) rRNA species as well as globin mRNA transcripts. The protocol is applicable to a wide range of RNA-Seq applications, including:
• gene expression analysis of high- and low-quality RNA samples;
• single nucleotide variation (SNV) discovery;
• splice junction and gene fusion identification; and
• characterization of both polyadenyated and nonpolyadenylated RNAs, including noncoding and immature RNAs.

Process Workflow


Library Construction Protocol

  1. Reagent Preparation
    This protocol takes 10 – 12 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 RNA-Seq Kit with RiboErase (HMR) Globin. 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 – 9.

    Volumes of additional reagents required for the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) Globin protocol are listed in Table 10.

    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 of 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 7B.

    Always ensure that the reagents required for oligo hybridization, rRNA/globin mRNA depletion, DNase digestion and the PEG/NaCl Solution are fully equilibrated to room temperature before use.

    Table 2. Oligo hybridization

    *If also processing non blood-derived samples, or if following the KAPA RiboErase (HMR) workflow, the volume of Globin Hybridization Oligos (HMR) must be replaced with an equal volume of RNase-free water (1 μL). For more information refer to the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) technical data sheet – KR1151 v5.17 (or later).

    Table 3. rRNA/globin mRNA depletion


    Table 4. DNase digestion


    Table 5. 1st strand synthesis


    Table 6. 2nd strand synthesis and marking


    Table 7A. A-tailing (uninterrupted protocol)


    Table 7B. A-tailing (safe stopping point)


    Table 8. Adapter ligation


    Table 9. Library amplification


    Table 10. Volumes of additional reagents required


  2. Oligo Hybridization and rRNA/globin mRNA Depletion
    This protocol requires 100 ng – 1 μg of total RNA, in 10 μL of RNase-free water.
    1. Ensure that the hybridization master mix (Table 2) and the depletion master mix (Table 3) are prepared and kept at room temperature before use.
    2. If also processing non blood-derived samples, or if following the KAPA RiboErase (HMR) workflow, the volume of Globin Hybridization Oligos (HMR) must be replaced with equal volume RNase-free water. For more information, refer to the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) technical data sheet – KR1151 v5.17 (or later).
    3. If rRNA and globin mRNA depletion efficiency will be assessed using qRT-PCR, a process control (no RNase H) will be required. Please refer to Appendix for more information.
    2.1
    Program a thermocycler as follows:


    2.2
    Assemble rRNA/Globin mRNA Hybridization reactions as follows:


    2.3
    Mix RNA and Hybridization master mix thoroughly by pipetting up and down at least 10 times using a mixing volume of 10 μL.
    2.4
    Place samples in the pre-programmed thermocycler and execute the program.
    2.5
    Ensure the depletion master mix containing RNase H is added while the samples are kept  at 45°C in a thermocycler. When the program reaches the pause step at 45°C, add the following to each 20 μL hybridization reaction and mix thoroughly by pipetting up and down at least 10 times using a mixing volume of 10 μL:


    2.6
    Resume the cycling program to continue with the depletion step (45°C for 30 min).
    2.7
    Proceed immediately to rRNA/Globin mRNA Depletion Cleanup (step 3).

  3. rRNA/Globin mRNA Depletion Cleanup
    3.1
    Perform a 2.2X bead-based cleanup by combining the following:


    3.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    3.3
    Incubate the plate/tube(s) at room temperature for 5 min to bind RNA to the beads.
    3.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    3.5
    Carefully remove and discard 75 μL of supernatant.
    3.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    3.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.8
    Carefully remove and discard the ethanol.
    3.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    3.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    3.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    3.12
    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.

  4. DNase Digestion
    To remove the hybridization oligonucleotides from the RNA/globin-depleted RNA, the sample is incubated with DNase. Ensure that the DNase digestion master mix (Table 4) is prepared and kept at room temperature.
    4.1
    Assemble DNase Digestion reactions as follows:


    4.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    4.3
    Incubate the plate/tube(s) at room temperature for 3 min to elute the RNA off the beads.
    4.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    4.5
    Carefully transfer 20 μL of supernatant into a new plate/tube(s). Discard the plate/tube(s) with beads.
    4.6
    Incubate the plate/tube(s) with supernatant using the following protocol:


    4.7
    Proceed immediately to DNase Digestion Cleanup (step 5).

  5. DNase Digestion Cleanup
    5.1
    Perform a 2.2X bead-based cleanup by combining the following:


    5.2
    Thoroughly resuspend the beads by pipetting up and down multiple times.
    5.3
    Incubate the plate/tube(s) at room temperature for 5 min to bind RNA 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 60 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.

  6. RNA Elution, Fragmentation and Priming
    Total RNA depleted of rRNA/globin mRNA is eluted from beads in Fragment, Prime and Elute Buffer (1X) and fragmented to the desired size by incubation at high temperature.

    Optional QC: If rRNA and globin mRNA depletion efficiency is going to be assessed using qRT-PCR, a process control (no RNase H) will be required and a modified RNA elution protocol followed. Please refer to Appendix for more information.
    6.1
    Prepare the required volume of Fragment, Prime and Elute Buffer (1X) by combining the following at room temperature:


    6.2
    Thoroughly resuspend the beads with purified, DNase-treated RNA in 22 μL of Fragment, Prime and Elute Buffer (1X) by pipetting up and down multiple times.
    6.3
    Incubate the plate/tube(s) at room temperature for 3 min to elute RNA off 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 transfer 20 μL of supernatant into a new plate/tube(s). Discard the plate/tube(s) with beads.
    SAFE STOPPING POINT
    Samples can be stored at -15°C to -25°C for ≤24hrs. When ready, proceed to step 6.6. 
    6.6
    Place the plate/tube(s) in a thermocycler and carry out the fragmentation and priming programas follows:

    6.7
    Place the plate/tube(s) on ice and proceed immediately to 1st Strand Synthesis (step 7).

  7. 1st Strand Synthesis
    7.1
    On ice, assemble the 1st strand synthesis reaction as follows:


    7.2
    Keeping the plate/tube(s) on ice, mix thoroughly by gently pipetting the reaction up and down several times.
    7.3
    Incubate the plate/tube(s) using the following protocol:


    7.4
    Place the plate/tube(s) on ice, and proceed immediately to 2nd Strand Synthesis and A-tailing (step 8).

  8. 2nd Strand Synthesis and A-tailing
    8.1
    On ice, assemble the 2nd strand synthesis and A-tailing reaction as follows:


    8.2
    Keeping the plate/tube(s) on ice, mix thoroughly by gently pipetting the reaction up and down several times.
    8.3
    Incubate the plate/tube(s) using the following protocol:


    8.4
    Proceed immediately to 2nd Strand Synthesis and Marking Cleanup (step 9).

  9. 2nd Strand Synthesis and Marking Cleanup
    9.1
    Perform a 1.8X 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 160 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
    Proceed immediately to A-tailing Immediately (step 10A), or follow the Safe Stopping Point instructions.
    SAFE STOPPING POINT
    Resuspend the beads in 15 μL A-Tailing Buffer (1X) (Table 7B), 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 10B).

  10. A-tailing
    A-tailing is performed either directly after 2nd Strand Synthesis and Marking Cleanup (step 9), 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 10A) or A-tailing after Safe Stopping Point (step 10B).

    10A. A-tailing Immediately
    10A.1
    Assemble the A-tailing reaction as follows:


    10A.2
    Mix thoroughly by pipetting up and down severaltimes.
    10A.3
    Incubate the plate/tube(s) using the following protocol:


    10A.4
    Proceed immediately to Adapter Ligation (step 11).

    10B. A-tailing after Safe Stopping Point
    10B.1
    To resume library preparation, combine the following reagents to perform A-tailing:


    10B.2
    Mix thoroughly by pipetting up and down several times.
    10B.3
    Incubate the plate/tube(s) using the following protocol:


    10B.4
    Proceed immediately to Adapter Ligation (step 11).

  11. Adapter Ligation
    11.1
    Dilute adapters in preparation for ligation targeting the following concentrations:


    11.2
    Set up the adapter ligation reactions as follows:


    11.3
    Mix thoroughly by pipetting up and down several times to resuspend the beads.
    11.4
    Incubate the plate/tube(s) at 20°C for 15 min.
    11.5
    Proceed immediately to 1st Post-ligation Cleanup (step 12). 

  12. 1st Post-ligation Cleanup
    12.1
    Perform a 1X bead-based cleanup by combining the following:


    12.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    12.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    12.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    12.5
    Carefully remove and discard 135 μL of supernatant.
    12.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    12.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    12.8
    Carefully remove and discard the ethanol.
    12.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    12.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    12.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    12.12
    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.
    12.13
    Remove the plate/tube(s) from the magnet.
    12.14
    Thoroughly resuspend the beads in 50 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    12.15
    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 13).

  13. 2nd Post-ligation Cleanup
    13.1
    Perform a 1X bead-based cleanup by combining the following:


    13.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    13.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    13.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    13.5
    Carefully remove and discard 95 μL of supernatant.
    13.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    13.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    13.8
    Carefully remove and discard the ethanol.
    13.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    13.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    13.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    13.12
    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.
    13.13
    Remove the plate/tube(s) from the magnet.
    13.14
    Thoroughly resuspend the beads in 22 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    13.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    13.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    13.17
    Transfer 20 μL of the clear supernatant to a new plate/tube(s) and proceed to Library Amplification (step 14).
    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 14). 

  14. Library Amplification
    14.1
    Assemble each library amplification reaction as follows:


    14.2
    Mix well by pipetting up and down several times.
    14.3
    Amplify the library using the following thermocycling profile:

    *Optimization of the annealing temperature may be required for nonstandard (i.e., other than Illumina TruSeq) adapter/primer combinations.

    14.4
    Proceed immediately to Library Amplification Cleanup (step 15).

  15. Library Amplification Cleanup
    15.1
    Perform a 1X bead-based cleanup by combining the following:


    15.2
    Mix thoroughly by vortexing and/or pipetting up and down multiple times.
    15.3
    Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind DNA to the beads.
    15.4
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    15.5
    Carefully remove and discard 95 μL of supernatant.
    15.6
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    15.7
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    15.8
    Carefully remove and discard the ethanol.
    15.9
    Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
    15.10
    Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
    15.11
    Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
    15.12
    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.
    15.13
    Remove the plate/tube(s) from the magnet.
    15.14
    Thoroughly resuspend the dried beads in 22 μL of 10 mM Tris-HCl (pH 8.0 – 8.5).
    15.15
    Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads.
    15.16
    Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
    15.17
    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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