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PROTOCOL FOR LIBRARY PREPARATION using KAPA RNA Hyper Prep Kit w/RiboErase    

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Procedure

400 ng DNase-treated RNA and a total of 9 cycles of PCR amplification of the library. The libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Kapa Biosystems). We followed the step-by-step protocol from the manufacturer (see Reference 2), attached using 400 ng DNase-treated RNA and a total of 9 cycles of PCR amplification of the library.

  1. Ensure samples meet following requirements
    RNA RIN >6

    RNA in 10 μl Elution Buffer

    25 ng < RNA < 1 μg

    # Samples: 12

  2. Oligo Hybridization and rRNA Depletion
    Hybridization MM
       1X
        12

    KAPA Hyb Buffer
        4
        52.8

    KAPA Hyb Oligo
        4
        52.8

    Water
        2     26.4
     <-- Keep MM at RT
    RNA
       10      --
    Total Volume
       20

    **Add 10 μl Hyb MM to RNA once Depletion MM is made.

    rRNA Depletion MM
                  1X
          12

    KAPA Depletion Buffer               3       39.6

    KAPA RNase H               2       26.4
          <-- Keep MM at RT
    RNA
                 20       --

    Total Volume
                 25
    **Add 5 μl MM to RNA once cycler at 45 °C. Pipette up/down.


    Thermal Cycler Conditions
    Temp
            Time
    1    Hybridization 95 °C
            2 min
    2 Ramp down to 45 °C at -0.1 °C/s


    3 Pause
    45 °C
            -
    4 Depletion
    45 °C
            30 min
    5 Hold
    4 °C
             -

  3. rRNA Depletion Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.

    2.2X KAPA Pure Bead
    1 Rxn (μl)
    Ribosomal-depleted RNA
    55
    KAPA Pure Beads
    55
    Total Volume
    80

    1. Mix samples by pipetting and incubate at RT for 5 min.
    2. Place tubes on magnetic stand and discard 75 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
      80% EtOH for 12 samples:
      4.8 ml 100% EtOH
      1.2 ml dH2O
      6.0 ml Total
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.

    **DO NOT OVERDRY.
    **Proceed immediately to next step.

  1. DNase Digestion
    DNase Digestion MM
    1X
    12

    KAPA DNase Buffer
    2.2
    29.0

    KAPA DNase
    2.0 26.4

    Water
    17.8
    235.0
    <-- Keep MM at RT
    Total Volume
    22


    **Add 22 μl MM to each tube, resuspend beads.

    1. Incubate beads at RT for 3 min.
    2. Place on magnetic stand and transfer 20 μl of supernatant to new tube.
      **Discard remaining beads.
    3. Incubate tubes at 37 °C for 30 min and hold at 4 °C.

  2. DNase Digestion Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.

    2.2X KAPA Pure Bead
    1x
    DNase-treated RNA
    20
    KAPA Pure Beads44 44
    Total Volume
    64

    1. Mix samples by pipetting and incubate at RT for 5 min.
    2. Place tubes on magnetic stand and discard 60 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.

    **DO NOT OVERDRY.
    **Proceed immediately to next step.

  1. RNA Elution, Fragmentation and Priming
    1X Fragment Buffer
        1X
        12

    RNase-free Water
        11
        145.2

    Fragment, Prime Elute Buffer
        11
        145.2
        <-- Keep at RT
    Total Volume
        22


    **Add 22 μl to each tube and resuspend beads.
    1. Incubate at RT for 3 min and place on magnetic stand.
    2. Transfer 20 μl of supernatant to new tube.
      **Discard tube with beads.
      **Safe stopping point: Store at -20 °C for 24 h.

    Input RNA Type
    Desired Size (bp)
    Fragmentation
    Intact
    100-200
    8 min at 94 °C

    200-300
    6 min at 94 °C

    300-400
    6 min at 85 °C
    Partially Degraded
    100-300
    1-6 min at 85 °C
    Degraded
    100-200
    1 min at 65 °C
    Place tubes on ice and proceed to next step.

  2. 1st Stand Synthesis
    1st Strand Synthesis 
                  1X
               12

    1st Strand Synthesis Buffer
                  31         
              372
    KAPA Script
                  2
              24
          <-- Keep on ice
    1st Strand Synthesis product
                  30            --
    Total Volume
                  60

    **Add 10 μl MM to each RNA sample.

    Mix samples with pipetting and incubate with following protocol:

    Thermal Cycler Conditions:
    Temp
           Time
    1   Primer extension
    25 °C
           10 min
    2 1st Strand Synthesis
    42 °C
           15 min
    3 Enzyme inactivation
    70 °C
           15 min
    4 Hold
     4 °C
            -
    **Place tube on ice and proceed immediately to next step.

  3. 2nd Strand Synthesis
    2nd Strand Synthesis 
            1X
            12

    2nd Strand Marking Buffer
            11
           132
    2nd Strand Synthesis & A tail Mix
            1
           12
             <-- Keep on ice
    Fragmented, Primed RNA
           20
    Total Volume
          30

    **Add 30 μl MM to each sample.

    Mix samples with pipetting and incubate with following protocol:

    Thermal Cycler Conditions:
    Temp
               Time
    1   2nd Strand Synthesis
    16 °C
              30 min
    2 A Tailing
    62 °C
              10 min
    3 Hold 4 °C            -
    **Place tube on ice and proceed immediately to next step.

  4. Adapter Ligation
    Adapter Ligation
    1X
               12

    Ligation Buffer
    40
              480

    DNA Ligase
    40
              120
       <-- Keep on ice
    Adapter stock (1.5 μM)
    5
               --

    2nd Strand Synthesis product
    60
               --

    Total Volume
    110


    **Add 45 μl MM to each sample.
    **Add adapter first then add mastermix.

    1. Mix samples on ice and incubate at 20 °C for 15 min.
    2. Proceed immediately to next step.

  5. 1st Post-Ligation Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.

    0.63X KAPA Pure Bead
    1X
    Adapter Ligated DNA
    110
    KAPA Pure Beads
    70
    Total Volume
    180
    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 175 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
      **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 50 μl 10 mM Tris-HCl, pH 8.0.
    7. Incubate beads at RT for 2 min.
      **Safe stopping point: Store at 4 °C for 24 h.

  6. 2nd Post-Ligation Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.

    0.7X KAPA Pure Bead
    1X

    Beads w/ Adapter-ligated DNA
    50

    20% PEG 8000/2.5 M NaCl Solution
    35
          <-- Equilibrate to RT, mix before use
    Total Volume
    85


    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 80 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
      **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 22.5 μl 10 mM Tris-HCl, pH 8.0.
    7. Incubate beads at RT for 2 min.
    8. Place on magnetic stand and transfer 20 μl supernatant to new tube.
      **Safe stopping point: Store at 4 °C for 1 wk or -20 °C for 1 mo.

  7. Library Amplification
    Library Amplification
    1X
            12

    KAPA HiFi HotStart Mix
    25
            330
             <-- Fully thaw and mix before use
    Library Amp Primer Mix
    5
            66
             <-- Keep on ice
    Purified, Adapter-ligated DNA
    20         --

    Total Volume
    50


    **Add 30 μl MM to each sample.


    Thermal Cycler Conditions:
    Temp
    Time

    1   Initial Denaturation
    98
    45 s
    2 Denaturation
    98
    15 s
    3 Annealing
    60
    30 s
    4 Extension
    72
    30 s
    5 Extension
    8 more times
            <-- Depends on quantity of starting material
    6 Final Extension
    72
    1 min
    7    Hold
    4 -
    Proceed immediately to next step.

  8. Library Amplification Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.

    1X KAPA Pure Bead
    1X
    Amplified library DNA
    50
    KAPA Pure Beads
    35
    Total Volume
    85

    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 82 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
      **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 22 μl 10 mM Tris-HCl, pH 8.
    7. Incubate beads at RT for 2 min.
    8. Place on magnetic stand and transfer 20 μl supernatant to new tube.
    9. Submit samples for QC/Seq.
    10. Store at 4 °C for < 1 week, or at -20 °C.

References

  1. Whipple, A. J., Jacobs, H. N., Breton-Provencher, V., Sur, M. and Sharp, P. A. (2019). Imprinted maternally-expressed microRNAs antagonize paternally-driven gene programs in neurons. bioRxiv: 717868.
  2. Kapa Biosystems. (2018). KAPA RNA HyperPrep Workflow: Recommendations and Expectations for RNA-sequencing Using Degraded Inputs. Kapa Biosystems. Wilmington, MA, USA, and Cape Town, South Africa.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:Amanda J. Whipple. (2019). PROTOCOL FOR LIBRARY PREPARATION using KAPA RNA Hyper Prep Kit w/RiboErase . Bio-101: e3497. DOI: 10.21769/BioProtoc.3497.
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