Extended Data Fig. 3: Characterizing massively parallel uORF reporters using flow cytometry.

a, Representative flow cytometry scatterplots of HEK 293-K^b cells transfected with the GFP mRNA reporter or pooled uORF reporters. Relative GFP and 25D1 fluorescence intensity between GFP and uORF-GFP reporters are shown in histograms as well as bar graphs. Error bars, mean ± s.e.m. n = 3 biological replicates. b, HEK293-K^b cells were transfected with mRNA reporters followed by FACS soring into GFP^H and 25D1^H populations. A bar plot (top) shows the ratio of triplet frequency within the random sequences enriched in the 25D1^H population over the GFP^H population. Only the top 10% sequence variants ranked in 25D1^H and GFP^H populations are used. The original frequency of triplets in different populations is shown as a heat map (bottom). c, Correlation of triplet frequencies within the sequence variants enriched in 25D1^H or GFP^H populations. All points are color-encoded based on the similarity to ATG.

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