Fig. 6.

Schematic demonstration of the custom designed 2-step RT protocol presented in this study. The cDNA synthesis is performed using an oligo-dT-adapter as RT primer and SMARTScribe™ Reverse Transcriptase (Takara Bio, Inc), which has an enhanced terminal nucleotidyl transferase (TdT)-like activity. After the full-length cDNA synthesis by incubation of the reaction mix at 42° for 70 min, a template-switching oligonucleotide (TSO) is added to the reaction mix, without removing the tubes from the thermal cycler. The reaction mix containing the TSO is further incubated at 42° for additional 20 min to facilitate the template-switching mechanism. Finally, thermal inactivation of the RT enzyme is achieved by incubating the RT mixture at 72 °C for 10 min