Extended Data Fig. 3. mRNA synthesis rate with MPRA.

(a) Measurements of mRNA synthesis rate with MPRA, experimental setup: Arabidopsis protoplasts were transformed with pTRP1-based libraries containing 12,000 fragments positioned downstream of the TSS and incubated for 5 h 40 min at room temperature with constant light. After addition of 5-ethynyl uridine (5-EU) and 20 min incubation²³, total RNA was extracted. Total mRNA was used for regular MPRA. Newly synthesized mRNA was isolated by click reactions followed by selection of biotinylated mRNA using beads. The isolated mRNA was used to generate MPRA libraries. The experiment was performed in two repeats. (b) Relative activity of downstream fragments inserted into pTRP1-based constructs as a function of the number of YVGATCBR consensus motifs, as in Fig. 3c. Group sizes: 6,855 (no motif), 956 (1 motif), 119 (2 motifs). Data represent average signal from two replicates of mRNA synthesis MPRA experiments for total mRNA and newly synthesized mRNA. Error bars represent the mean ± 1 standard deviation.