Sequential optimization of template switching activity of MarathonRT. (A) Outline of the experimental design to monitor the template-switching activity. The figure describes each step involved in the template-switching reaction of MarathonRT, including nontemplated “AAA” addition, annealing between the “AAA” overhang and 3′-UUU of an RNA TSO, and primer extension continuing on the TSO. (B) A gel figure showing the template switching products of MarathonRT under representative reaction conditions, including the original RT buffer with an equimolar mix of 0.5 mM dATP, dCTP, dGTP, and dTTP (lane 1), the same reaction buffer as lane 1 but using a dNTP mix containing 0.8 mM dATP and 0.4 mM dCTP, dGTP, and dTTP (lane 2), the same reaction condition as lane 2 with additional 12% PEG4000 (lane 3), reduced KCl concentration from 200 mM to 150 mM compared to lane 3 (lane 4), and further reduced KCl concentration to 100 mM (lane 5). (C) Quantification of template switching efficiency for each condition in (B). Each reaction was repeated three times with mean and standard deviation shown in the figures. P-values were calculated using unpaired one-tailed Student's t-test.
