Extended Data Fig. 2: Comparison between Ribo-ITP and conventional ribosome profiling.

Ribo-ITP experiments from ~100-cells were compared to the conventional ribosome profiling from ~10M cells for all panels. a, Transcripts with at least one count per million (cpm) in at least two out of three replicates were selected. Log₂ of mean cpm (x-axis) was plotted against the square root of the standard deviation of cpm (y-axis). Green (Ribo-ITP) and red (conventional) points represent individual transcripts with mean log₂(cpm) > 2. b, The percentage of clipped reads that mapped to ribosomal RNAs was calculated using RiboFlow⁶¹. The mean and its standard error were shown (n = 3; see also Supplementary Table 1). c, In the metagene plots, position 0 corresponds to the start (left, light green) or stop (right, dark green) site. Ribosome footprints were adjusted according to their A-site offsets. One representative replicate for each method is plotted. d, The mean percentage of specific read lengths among the total mapped reads is plotted. Ribbons around the lines represent standard error of the mean. e, The percentage of ribosome profiling reads mapped to the CDS is indicated for each experiment (left). Single cell data from K562 cells were generated using either RNase I digestion (1-1, 1-2, 1-3) or MNase (1-4, 1-5). The sum of weighted counts across transcripts were plotted for comparison (right). The weighted sum was calculated for each transcript by multiplying its region length by the total number of ribosome footprints. f, Ribosome footprints from Ribo-ITP and the conventional method were used to calculate the mean fraction of reads mapping to each reading frame (Methods). Error bars indicate the standard error of the mean of replicates.