NGS RNA First Strand Synthesis Module

The NGS First Strand Synthesis Module contains the enzymes and buffers required to convert RNA into cDNA using random priming. The fast, user-friendly workflow has minimal hands-on time and is compatible with upstream poly(A) mRNA enrichment and rRNA depletion methods; it is also compatible with downstream 2nd strand cDNA synthesis for RNA-seq workflows.

Applications

• cDNA synthesis for RNA library preparation and sequencing.

Protocol 

1.     In a nuclease-free 0.2 mL PCR tube, add 10 μL of fragmented mRNA and 1 μL of Random Primers.

2.     Incubate in a preheated thermocycler for 5 minutes at 65°C with heated lid set to 100°C. Hold at 4°C.

3.     Spin tube briefly and place on ice.

4.     To the fragmented mRNA and Random Primers add:

         First Strand Synthesis Reaction Buffer            4 μL
         First Strand Synthesis Enzyme Mix                 1 μL
         Nuclease-free Water                                        4 μL
        Total volumes                                                   20 μL

5.     Mix by pipetting gently up and down.

6.     Incubate the samples in a preheated thermal cycler (with the heated lid set to 100°C):

       10 minutes at 25°C
       30 minutes at 68°C
       Hold at 4°C

7.     Place the tube on ice.

8.     Proceed directly to second strand synthesis using NGS RNA Second Strand Synthesis Module (Cat no. E118).