Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

IRD700 fluorescent 5 -labeled oligonucleotides were from IDT DNA. The standard limited primer extension reaction was in 12.5 l volume with 1× Thermo buffer (Biolabs), 12 nM of a specific IRD700-labeled fluorescent primer (IDT-DNA, Supplementary Table S1), a mix of 1 M of three dNTPs with the missing dNTP replaced by the corresponding terminator chain reaction acydNTP (Biolabs) at 50 M, and 0.6 unit of Vent exo-polymerase (Biolabs). The quantity of (RT)-PCR template was about three times lower than that of the fluorescent primer. On average each primer molecule is utilized on 20 occasions for chain extension during the 60-cycle PCR reactions. The PCR cycle was: denaturation at 94 • C for 2 min, then 60 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s and elongation at 72 • C for 30 s. The final elongation was at 72 • C for 2 min. In all cases each RT reaction and its subsequent analysis was repeated at least twice. Reaction products were analyzed on 15% sequencing gels. Image capture was performed with a LiCor Sequencer.