Extended Data Fig. 3: Development of dpCoA-TLC.
a, HPLC chromatograms showing complete hydrolysis of dpCoA by AtNUDT11, followed by the production of new dpCoA with the addition of PPAT and ATP. b, TLC analysis to evaluate of the effectiveness of the NAP-10 column in removing free dpCoA from total RNA for dpCoA-TLC assays. Total RNA (500 μg) was incubated with 10 nmol or 100 nmol of [α-32P]-dpCoA and then processed using the NAP-10 column. The [α-32P]-dpCoA signal was visualized with the Typhoon phosphorimager. c, TLC analysis confirming the migration position of [α-32P]-AMP. Rpp refers to RNA 5′ polyphosphatase, which hydrolyzes [α-32P]-ATP to [α-32P]-AMP. d, Detection of dpCoA-RNA in E. coli DH5α total RNA, isolated from stationary phase by dpCoA-TLC. The signal of [α-32P]-dpCoA was visualized with a Typhoon phosphorimager. e, Agarose gel electrophoresis showing the separation of long (>200 nt) and short RNAs (<200 nt) from Arabidopsis seedlings. Data are representative of three independent experiments. f, dpCoA-RNA in long RNA and short RNA, isolated from Arabidopsis seedlings, was analyzed using dpCoA-TLC. The [α-32P]-dpCoA cap signal was visualized with the Typhoon phosphorimager, and the signal intensities were measured using ImageJ. g, Bar plot displaying the quantification of dpCoA-RNA in f. The amount of dpCoA-RNA was calculated using a calibration curve generated from a concentration series of the dpCoA standard in f. Error bars represent the mean ± s.e.m. from three independent biological replicates. Statistical significance was determined using a two-tailed Student′s t-test.
