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  • Library Preparation (E7580)

    Introduction

    Libraries prepared by this method are compatible with paired-end flow cells.

    Starting Material: 100 ng–1 μg Total RNA.

    Protocol

    Ligate the Multiplex 3´ SR Adaptor

    Note: For total RNA inputs of 100 ng, dilute the 3´ SR Adaptor for Illumina 1:2 in nuclease free water.
    1. Mix the following components in a sterile nuclease-free PCR tube:
      Input RNA   1–6 μl
      3´ SR Adaptor for Illumina  1 μl
      Nuclease-Free Water   variable
      ---------------------------------------------------------
      Total volume   7 μl

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

    3. Add the following Components:
      3´ Ligation Reaction Buffer (2X)   10 μl
      3´ Ligation Enzyme Mix   3 μl
      ---------------------------------------------------------
      Total volume   20 μl

    4. Incubate for 1 hour at 25°C in a thermal cycler.

      Note: Longer incubation times and reduced temperatures (18 hours; 16°C) increase ligation efficiency of methylated RNAs such as piwi-interacting RNAs (piRNAs) (if present in the sample). However, some concatamerization products might be formed.

    Hybridize the Reverse Transcription Primer

    This step is important to prevent adaptor-dimer formation. The SR RT Primer hybridizes to the excess of 3´ SR Adaptor (that remains free after the 3´ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step.

    Note: For total RNA inputs of 100 ng, dilute the SR RT Primer for Illumina 1:2 in nuclease free water.
    1. Add the following components to the ligation mixture from step 4 and mix well:
      Nuclease-Free Water   4.5 μl
      SR RT Primer for Illumina  1 μl
      ---------------------------------------------------------
      Total volume now should be   25.5 μl

      Note: For total RNA inputs less than 5 μg, dilute (1:2 dilution in Nuclease- Free Water) the Multiplex SR RT Primer.

    2. Heat samples for 5 minutes at 75°C. Transfer to 37°C for 15 minutes, followed by 15 minutes at 25°C.
    Ligate the 5´ SR Adaptor

    1. With 5 minutes remaining, resuspend the 5´ SR adaptor in 120 μl of nuclease free water.

      Note: For total RNA inputs of 100 ng, dilute the 5´ SR Adaptor for Illumina 1:2 in nuclease free water.

    2. Aliquot 1.1 N μl of the 5´ SR Adaptor into a separate, nuclease-free 200 μl PCR tube, with N equal to the number of samples being processed for the current experiment.

    3. Incubate the adaptor in the thermal cycler at 70°C for 2 minutes and then immediately place the tube on ice. Keep the tube on ice and use the denatured adaptor within 30 minutes of denaturation.

      Note: Store the remaining resuspended 5´ SR adaptor at -80°C.

    4. Add the following components to the ligation mixture from step 6 and mix well:
      5´ SR Adaptor for Illumina (denatured) 1 μl
      5´ Ligation Reaction Buffer (10X)   1 μl
      5´ Ligase Enzyme Mix   2.5 μl
      ---------------------------------------------------------
      Total volume   30 μl

    5. Incubate for 1 hour at 25°C in a thermal cycler.

    Perform Reverse Transcription

    1. Mix the following components in a sterile, nuclease-free tube:
      Adaptor Ligated RNA from Step 11   30 μl
      First Strand Synthesis Reaction Buffer  8 μl
      Murine RNase Inhibitor   1 μl
      ProtoScript II Reverse Transcriptase   1 μl
      ---------------------------------------------------------
      Total volume   40 μl

    2. Incubate for 60 minutes at 50°C.

    3. Immediately proceed to PCR amplification.

      Safe Stopping Point: If you do not plan to proceed immediately to PCR amplification, then heat inactivate the RT reaction at 70°C for 15 minutes. Samples can be safely stored at -15°C to -25°C.

    Perform PCR Amplification

    1. Add the following components to the RT reaction mix from step 14 and mix well:
      LongAmp Taq 2X Master Mix   50 μl
      SR Primer for Illumina   2.5 μl
      Index (X) Primer*   2.5 μl
      Nuclease free water   5 μl
      Total volume now should be   100 μl

      *Note: The NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 2) contains 13-24 PCR primers, each with a different index. For each reaction, only one of the 12 PCR primer indices is used during the PCR step.

      PCR Cycling conditions:

      CYCLE STEP TEMP TIME CYCLES
      Initial Denaturation 94°C 30 sec 1
      Denaturation
      Annealing
      Extension
      94°C
      62°C
      70°C
      15 sec
      30 sec
      15 sec
      12-15*
      Final Extension 70°C 5 min 1
      Hold 4°C  
      *Amplification conditions may vary based on RNA input amount, tissue, and species. This protocol was optimized using 1 μg of total RNA from human brain and 12 PCR cycles. The number of PCR cycles can be adjusted if clear and distinct bands are not observed in the gel image. Run only between 12 and 15 cycles. For 100 ng total RNA input run 15 cycles of PCR.