Reverse transcription and second strand synthesis

For cDNA synthesis, 2.5 μM of a random primer with a known 20-nt tag sequence (SISPA-N, 5’-GCT GGA GCT CTG CAG TCA TCN NNN NN-3’) was added to the nucleic acid samples prepared in the step above, incubated at 97°C for 3 min, and cooled on ice. Then 20 μl of cDNA-mix (RevertAid First Strand H minus cDNA Synthesis kit; Thermo Fisher, Waltham, Massachusetts, USA), consisting of 10 μl of 5 X reaction buffer, 5 μl of 10 mM dNTP mix, 2.5 μl of 20U/ μl Ribolock RNase inhibitor, and 2.5 μl of 200U/ μl RevertAid H minus RT, was added to each sample, and the suspension was incubated for 10 min at 25°C, 90 min at 45°C, and 5 min at 70°C. Finally, 1 μl of RNase H (New England Biolabs, Ipswich, Massachusetts, USA) was added to degrade residual RNA, and the sample was incubated for 20 min at 37°C. For second strand synthesis, 45.5 μl of cDNA, 0.6 μl of 10 X Klenow buffer (Thermo Fisher, Waltham, Massachusetts, USA), 0.4 μl of 100 μM SISPA-N primer, and 1 μl of 10 mM dNTP were mixed, denatured for 1 min at 95°C and cooled on ice. Then, 2.5 μl of Klenow Fragment 3’→5’ exo- (Thermo Fisher, Waltham, Massachusetts, USA) was added, and the mixture was incubated for 15 min at 25°C and for 1 h at 37°C and subsequently denatured for 1 min at 95°C. Following cooling on ice, another 1.25 μl of Klenow Fragment 3’→5’ exo- was added and the reaction was allowed to proceed for 15 min at 25°C and 1 h at 37°C. Samples were purified using the PureLink® PCR Micro kit (Invitrogen, Thermo Fisher, Waltham, Massachusetts, USA) according to the manufacturer’s protocol. Finally, dsDNA was eluted with 12 μl of buffer E1 (Invitrogen, Thermo Fisher, Waltham, Massachusetts, USA) [64].