Fig. 1: DualRP allows for the study of ribosome occupancy and tRNA-Ribosome association in two interacting cell types.

a Schematic diagram of dual ribosome profiling (Dual-RP): Ribosomes from two distinct cell types are individually tagged with chimera proteins, eGFP-RPL10a and mCherry-RPL10a. The two cell populations are mixed, lysed, and ribosomes from each cell type are then immunoprecipitated using highly specific nanobodies. Subsequently, libraries for next-generation sequencing are prepared from the recovered ribosome-protected fragments (RPFs). b Immunoprecipitation experiments with beads coated with nanobodies against GFP (IP: GFP) or against mCherry (IP: mCherry), followed by western blot analysis in SUM-159PT-GFP-RPL10a and MRC5-mCherry-RPL10a cells. The blots are representative of 3 independent experiments. c Principal Component Analysis (PCA) from ribosome profiling libraries generated from pull-down of mono- and co-cultures of SUM-159PT-GFP-RPL10a and MRC5-mCherry-RPL10a. Mixes of lysed mono-cultures were used as controls (Post-lysis control). d tRNA–ribosome association assay in SUM-159PT-GFP-RPL10a and MRC5-mCherry-RPL10a cells cultured under serine/glycine starvation. Shading within the graph indicates decreasing serine/glycine concentrations, ranging from 0.4 mM to 0 mM. PD pull-down. Data are presented as mean ± SD (n = 3). e Normalized cell numbers at different serine/glycine concentrations. The data are expressed as relative cell numbers compared to the untreated group at the endpoint. Measurements were taken 48 hours after plating. Data represent mean ± SD from biologically independent experiments (n = 3).