Selenium-modified deoxythymidine triphosphates reverse transcription and ligation assisted PCR analysis (SLAP) quantifies the modification frequency on PAN RNA. (A) Native PAGE shows PCR products derived from the modified (100% m6A) and unmodified (0% m6A) RNA standards, combined at the indicated percentages and subjected to SLAP analysis. The negative (−) and positive (+) reactions are indicated on top, together with the percentage of modified RNA. (B) The graph represents the SLAP standard calibration curve created based on quantification of results from panel A. The x-axis represents m6A fractions tested, while the y-axis represents corresponding densitometric measurements (n = 3). (C) Native PAGE for representative replicates (n = 3) of the SLAP analysis performed for nt 18, 203, 672, 1041, and 1048 on PAN RNA. Negative controls for the SLAP analysis include unmodified adenines on PAN RNA at nt 366 and 410 (n = 3). The time points of infection are indicated on top (0–72 h pi). The position of products specific for modified and unmodified sites is labeled. L stands for 50 bp DNA ladder. (D) The column graphs represent the average modification frequency on PAN at each site and the time point of infection (n = 3). Standard deviations for frequency measurements are indicated.
