Fig. 1: Analysis of CHO cell translation using ribosome footprint profiling.
From: Detection of host cell microprotein impurities in antibody drug products
a 8 replicate shake flasks were seeded with a mAb-producing CHO-K1 cell line cultured for 48 h; at this point, the temperature of 4 shake flasks was reduced to 31 °C. At 72 h post-seeding, cells were harvested from the non-temperature and temperature-shifted cultures. We utilised (b) Ribo-seq using different inhibitors to capture information from initiating (harringtonine) and elongating (cycloheximide and no drug) ribosomes. The chemical structures shown for harringtonine (CID = 276389) and cycloheximide (CID = 6197) were obtained from PubChem94. In addition, (c) RNA-seq was used to measure the RNA levels. Following pre-processing of the raw Ribo-seq data, we (d) retained reads within the expected size range (coloured in blue) of RPFs (28-31nt). As expected, no peak was observed for the RNA-seq data. An optimum P-site offset of 12 nt was selected for all datasets, where (e) an average of 66% of RPFs exhibited triplet periodicity. No framing pattern was observed for the RNA-seq data. A metagene analysis was conducted for each Ribo-seq dataset, confirming (f) the expected enrichment of RPFs at the TIS of annotated protein-coding genes for harringtonine Ribo-seq when compared to elongation Ribo-seq (average cycloheximide and no-drug treated RPFs). Separate metagene profiles for CHX and ND are shown in Supplementary Fig. 3. Source data are provided as a Source Data file. Panels a, b and c created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
