Figure 1.

Ribosome footprinting of primary cultures is reproducible. (A) Illustration of the experimental design. Primary cultures were derived from mouse brains, matured for 7 days, then replicate cultures were exposed to depolarizing stimuli. Parallel RNAseq (not shown) and RF were conducted to measure transcript abundance and ribosomal occupancy of mRNA, respectively. RF entails harvesting cycloheximide (CHX)-stalled ribosomes, then digesting with RNAse I all mRNA except fragments physically protected by ribosomes. Libraries were prepared from protected fragments and aligned to the transcriptome, enabling downstream computational analyses. (B,C) 7-day primary cultures containing a mixture of neurons and glia. (B) Immunostaining for NeuN confirms the presence of neurons (white arrows) as well as non-neuronal cells (yellow arrowheads). (Blue is DAPI staining for nuclei; Scale bar = 17 μM) (C) Double immunostaining for Aqp4 and GFAP reveals astrocytes (white arrows) and putative non-glial cells (yellow arrowheads). (Blue is DAPI staining for nuclei; Scale bar = 70 μM). (D) Screenshot of RNASeq read depth in counts per million (CPM), showing fragments covering the entirety of a representative mRNA (bottom), and RF read depth, showing coverage only of CDS and 5'UTR (top) using Map2 gene. (E–G) Scatterplots comparing the replicate cultures show high levels of reproducibility for (E) transcript levels, (F) RF densities as well as (G) TE in both untreated (gray) and KCl-treated samples (red) (all Pearson r > 0.96).