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  • Library Preparation Using NEBNext® Multiplex Small RNA Library Prep Set for SOLiD™ (Set 1) (E7450)

    Symbols

         This is a point where you can safely stop the protocol and store the samples at -20°C for up to 72 hours.
         This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

    Starting Material: 1–10 μg total RNA. Use half of the listed volume for the following reagents if your input is < 5 μg.
    • (green cap) 3´ SR Adapter 3
    • (pink cap) SR RT Primer 3
    • (yellow cap)  5´ SR Adapter 3
    It is preferable that all reactions be done in strip tubes and incubated on a thermalcycler.


    Ligation of 3´ and 5´ Adaptors (~1 hour and 15 minutes to the first safe stop point)

    1. Mix the following components in sterile nuclease-free strip tubes:
      Input RNA    1–6 μl
      NEBNext 3´ SR Adaptor 3    1 μl
      Nuclease-free Water    variable 
      ------------------------------------------------------------------
      Total volume   7 μl

    2. Incubate in a preheated thermal cycler at 70°C for 2 minutes and transfer tube to ice.

    3. Add the following components:
      3´ Ligation Reaction Buffer    10 μl
      3´ Ligation Enzyme Mix    3 μl
      ------------------------------------------------------------------
      Total volume    20 μl

    4. Incubate at 25°C for 1 hour in a thermal cycler.  The sample can be safely stored at -20°C for 72 hours at this point. ~1 hours and 15 minutes to the next safe stop point.

    5. Add the following components to the ligation mixture from step 4 and mix well:
      Nuclease-free Water    4.5 μl
      SR RT Primer 3    1 μl
      ------------------------------------------------------------------
      Total volume    25.5 μl

    6. Heat samples at 75°C for 3 minutes then ramp to 4°C at a rate of 0.3°C/sec and transfer to ice.

    7. Resuspend the 5´ SR adaptor 3 in 35 μl Nuclease-free Water. Heat  the resuspended adaptor at 70°C for 2 minutes and transfer to ice (only heat  denature once).

    8. Add the following components to the ligation mixture from step 7 and mix well:
      5´ SR Adaptor 3 (from Step 7)    1 μl
      5´ Ligation Reaction Buffer    1 μl
      5´ Ligation Enzyme Mix    2.5 μl
      ------------------------------------------------------------------
      Total volume    30 μl
    9. Incubate at 25°C for 1 hour in a thermal cycler.  The sample can be safely stored at -20°C for 72 hours at this point. ~1 hour and 30 minutes to the next safe stop point.

    Reverse Transcription

    1.  Mix the following components in sterile nuclease-free strip tubes:
      3´→5´ Ligated RNA from step 9    14 μl
      NEBNext First Strand Synthesis Reaction Buffer    4 μl
      Murine RNase Inhibitor    1 μl
      ------------------------------------------------------------------
      Total volume    19 μl

    2. Heat the sample at 42°C for 2 minutes and then add the following component.
      Preheated RT buffer and sample mix    19 μl
      ProtoScript II Reverse Transcriptase    1 μl
      ------------------------------------------------------------------
      Total volume    20 μl
    3. Incubate mixture for 42°C for 1 hour and then at 70°C for 15 minutes.  The sample can be safely stored at -20°C for 72 hours at this point.

    PCR Amplification

    1. Mix the following components in sterile strip tubes:
      RT reaction mixture (from step 12)    20 μl
      OneTaq Hot Start 2X Master Mix    25 μl
      Index Primer (X)*    2.5 μl
      SR Primer R3    2.5 μl
      Total volume    50 μl
      ------------------------------------------------------------------
      * Note: The kit contains 16 index primers, each with a unique index. For each reaction, only one Index Primer is used.

    2. PCR cycling conditions:
      CYCLE STEP TEMP TIME CYCLES
      Initial Denaturation 94°C 30 sec 1
      Denaturation
      Annealing
      Extension
      94°C
      60°C
      65°C
      10 sec
      30 sec
      15 sec
      12-15
      Final Extension 65°C 5 min 1
      Hold 4°C  

    Size Selection of Amplified cDNA Library

    1. Add 10 μl of Gel Loading Dye, Blue (6X) to each amplified cDNA construct (60 μl total volume).

    2. Load 5 μl of Quick-Load pBR322 DNA-MspI Digest in one well on a 6% PAGE gel.

    3. Load each amplified cDNA construct with loading dye by splitting into two wells (30 μl each) on the 6% PAGE gel.
    4. Run the gel at 120 V until the front of the dye reaches the bottom of the gel (~60 minutes). Do not let the dye exit the gel.

    5. Remove the gel from the apparatus and stain with SYBR Gold nucleic acid gel stain in a clean container for 10 minutes on orbital shaker and view the gel on a UV transiluminator.

    6. Cut the bands corresponding to ~110–119 bp, which correspond to adaptor-ligated constructs derived from the 21 and 30 nucleotide RNA fragments, respectively. DO NOT cut the 89 bp band out, as this is adaptor dimer (Figure 1). 

      E7450
      Figure 1: Small RNA Libraries
      Small RNA library generated from 5 μg Human Brain Total RNA (Lane 2) and 5 μl of the Quick-Load pBR322 DNA-MspI Digest (Lane 1). The 114 nucleotide band in Lane 2 contains prepared miRNA library generated from ~21 nucleotide small RNA fragments. The 93 nucleotide band in Lane 2 contains adapter dimer

    Gel Purification of Amplified cDNA Library

    1. Place the gel slice in a 1.5 ml tube and crush the gel slice with the RNase-free Disposable Pellet Pestles and soak in 100 μl DNA Gel Elution Buffer (1X).

    2. Rotate for 2–18 hours at room temperature.

    3. Transfer the eluate and the gel debris to the top of a gel filtration column, and centrifuge the filter for 2 minutes at 14,000 rpm.

    4. Check the size (should be between 110-130 bp), purity and concentration on an Agilent 2100 Bioanalyzer® (Agilent Technologies, Inc.) using a DNA high sensitivity on a DNA 1000 CHiP. If the gel doesn't run properly for your sample perform the following ethanol precipitation.

    5. Recover eluate, add 1 μl Linear Acrylamide, 25 μl 3M sodium acetate pH 5.2 and 750 μl of 100% ethanol and vortex well.

    6. Precipitate in a dry ice/methanol bath for at least 30 minutes then spin in a microcentrifuge (>14.000 x g) for 30 minutes at 4°C.

    7. Remove the supernatant taking care not to disturb/remove the pellet and wash the pellet with 80% ethanol by vortexing vigorously.

    8. Spin in a microcentrifuge (>14.000 x g) for 30 minutes at 4°C.

    9. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.

    10. Resuspend pellet in 10 μl TE Buffer. Perform the following quality control analysis on your sample library to quantify the DNA concentration.

    11. Load 1 μl of the reconstituted construct on an Agilent 2100 Bioanalyzer using a DNA High Sensitivity or an Agilent DNA-1000 chip (Figure 2).

    12. Check the size (should be ~110–119 bp), purity and concentration of the sample. The final product should be a distinct band. If you see undesirable peaks (bigger or smaller than your expected range sizes) perform a second round of size selection.

      Figure 2: Agilent Bioanalyzer Trace of a final Human Brain miRNA Library showing a 126 nM peak peak between 114 and 123 bp.