Protocol for Use with Previously Fragmented RNA - NEBNext mRNA First Strand Synthesis Module (E7525)

Protocol B for Use with Previously Fragmented RNA

Starting Material: 100–250 ng of purified mRNA fragmented to 200 nt and cleaned up in 13.5 µl of nuclease free water

1. First Strand cDNA Synthesis (Non-Directional Reaction Setup)
   Mix the following components in a sterile PCR tube
   

   Component	Volume
   Fragmented mRNA	13.5 µl
   (pink) Random Primers	1 µl
   Total Volume	14.5 µl

2. Incubate in a preheated thermocycler for 5 minutes at 65°C with heated lid set to 105°C. Hold at 4°C.
3. Spin tube briefly and place on ice 8
4. To the fragmented mRNA and Random Primers add:
   

   Component	Volume
   (pink) First Strand Synthesis Reaction Buffer	4 µl
   (pink) Murine RNase Inhibitor	0.5 µl
   Total Volume	19 µl

5. Incubate in a preheated thermocycler for 2 minutes at 25°C.
6. Add 1 µl ProtoScript II Reverse Transcriptase to the reaction.
7. Incubate the samples in a preheated thermal cycler (with the heated lid set to ≥ 80°C)
   10 minutes at 25°C
   50 minutes at 42°C
   15 minutes at 70°C
   Hold at 4°C
8. Place the tube on ice.
9. Proceed directly to second strand synthesis using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111).