Supplementary Fig. 10: Selection of stalling sequences from mRNA libraries.

a, Schematic of mRNA library designs, with randomized locations in the CDH1 stalling sequence indicated. Numbers indicate amino acids in the CDH1 sequence. b, Protocol for mRNA library selections using in vitro translation reactions. RNase H treatment was carried out first during RNC binding and purification on anti-FLAG beads, and a second time after RNC release from the beads. c, Luciferase activity of translation reactions with luciferase reporters containing the CDH1 stalling sequence with the WT or RSCK motif near the PTC, as a function of PF846 concentration (data represent mean ± s.d., n = 3 independent experiments). Source data for panel c are available in Supplementary Data Set 3. d, Western blot of affinity-purified stalled CDH1-derived nascent chains. Reactions in the absence or presence of 50 µM PF846 are shown. Western blot of eRF1 for each sample is also shown, along with a Western of RPS19 as a loading control. e, MA plot of sequences enriched for PF846-induced translation elongation stalling, compared to translation reactions in the absence of PF846. Sequences were enriched from the mRNA library of CDH1-derived nascent chains with four amino acid positions randomized near the predicted location of PF846 binding in the ribosome exit tunnel. Log₂-fold enrichment of sequences is plotted against total read count, for experiments carried out in duplicate. No sequences were enriched with an adjusted P-value < 0.01. Significance was tested by DESeq2 using the Wald test (two-sided) and adjusted for multiple comparisons by the Benjamini-Hochberg method³⁹.