2 nd Strand cDNA Synthesis Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)

The following protocol can be used to synthesize ds cDNA that covers the full 5′ transcription start site when the 5′ sequence of the transcript is unknown, providing an advantage over the Gubler and Hoffman 2 nd Strand cDNA Synthesis method [1, 2]. This protocol contains two steps. In the first step, template switching reverse transcription generates cDNAs with a universal sequence of choice attached to the 3′ end of cDNA, mediated by the template switching oligo (TSO). In the second step, RNA is hydrolyzed and 2 nd strand cDNA is subsequently synthesized by primer extension using the TSO as a primer.

Sample Recommendations:

To synthesize double stranded cDNA containing transcription start sites high quality intact RNA is required, usually in the range of 100 ng - 5 µg. The RNA sample should be free of salts (e.g., Mg 2+ , guanidinium), divalent cation chelating agents (e.g., EDTA, EGTA, citrate) or organics (e.g., phenol, ethanol). If an excess amount of genomic DNA is present in RNA samples, an optional DNase I treatment can be performed. Inactivate or remove DNase I after treatment.

Reaction Preparation

1. Briefly centrifuge the Template Switching RT Enzyme Mix to collect the solution to the bottom of the tube, then place on ice.
2. Thaw the Template Switching RT Buffer at room temperature completely. Vortex and centrifuge briefly to collect the solution to the bottom of the tube, then place on ice.

cDNA Synthesis and Template Switching

Please note that the volume needed for each reagent is based on a final cDNA Synthesis and Template Switching reaction volume of 10 μl. If desired, the reaction can be scaled up proportionally, while observing the RNA input amount limit, to a final volume of 20 μl.

Primer Annealing for 1st Strand Synthesis

1.1 To anneal the RT primer with the RNA templates, in a 0.2 ml nuclease free PCR tube, prepare the reaction as follows (on ice):