cDNA Conversion

cDNA stands for complementary DNA, and can be described as a double stranded DNA that is made with RNA as template. Our ultimate goal is the sequence the RNA that we have extracted - and with our sequencing method of choice, we need to turn the RNA back into DNA for it to be read properly. To do this, we use a technique known as reverse transcription to read and copy the RNA contents onto a newly made DNA strand, with no genetic information lost!

Experiment

Reverse transcription consists of 3 major steps - complementary DNA strand synthesis, RNA digestion and synthesis of second strand. The steps are described below: Synthesis of complementary DNA strand:
Due to previous polyA addition to 3´OH, all RNA molecules have similar sequence at 3´ end which only differs in number of added adenine bases. This allows using polyT (VNP) primers (Oxford Nanopore) to anneal to RNA template and reverse transcriptase (SuperScript IV, ThermoFisher) can initiate the transcription.

A second, so-called strand switching primer is added to the reaction. This compensates for under-representations of 5´ends in cDNA by introducing an additional template and therefore protecting the terminal base pairs. Terminal transferase activity of the RT adds a number of deoxycytidine bases. The SSP primer is complementary to these bases and acts as an extended template for the RT, not only protecting the terminal bases, but also allowing to introduce sequence of choice into the newly synthesized first strand.

RNA template digestion:
RNA template needs to be removed before the second DNA strand can be synthesized. This is done by adding ribonucleases (RNAse Cocktail Enzyme Mix, ThermoFischer) into the reaction and incubating. The enzyme mix consists of RNase A and T1.

Second strand synthesis:
The second DNA strand is synthesized using LongAmp Taq Polymerase (NEB) incubated for one round. Primers used in the reaction are complementary to the sequences introduced by SSP and VNP primers.