NEBNext Multiplex Small RNA Sample Prep Set for Illumina - Library Preparation (E7330)

Introduction

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

Starting Material: 100 ng–1 μg Total RNA.

Protocol

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.

5. 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
   
   
6. Heat samples for 5 minutes at 75°C. Transfer to 37°C for 15 minutes, followed by 15 minutes at 25°C.

7. 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.

8. 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.

9. 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 Multiplex 5´ SR adaptor at -80°C.

10. 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

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

12. 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

13. Incubate for 60 minutes at 50°C.

14. 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.

15. 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
    
    PCR Cycling conditions:

    CYCLE STEP	TEMP	TIME	CYCLES
    Initial Denatuation	94°C	30 seconds	1
    Denaturation Annealing Extension	94°C 62°C 70°C	15 seconds 30 seconds 15 seconds	12-15*
    Final Extension	70°C	5 minutes	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 using 12 PCR cycles. The number of PCR cycles can be adjusted if clear and distinct bands are not observed in the gel image. Run between 12 and 15 cycles. For 100 ng total RNA input run 15 cycles of PCR.