Effective extraction of polyribosomes from primary astrocytes exposes strategies of gene expression regulation [RNAseq_Riboseq]

We optimized a standard polysome profiling protocol considering the unique morphology and ribosomal distribution of astrocytes. This 'astrocyte-optimized' protocol was then used to study primary astrocytes treated with proinflammatory cytokines, enabeling Ribo-seq analysis for measurment of the transcriptome, translatome, and translation efficiency with a coverage of over 12,000 genes. We found several regulation strategies of gene expression executed as part of an extensive inflamatory response. Enhanced translation efficiency was one of these strategies, used specifically for genes related to oxidative phosphorylation, and ribosomal proteins. Overall design: Three biological repetitions of primary astrocytes were treated or not with 20ng/ml TNFa and 0.25ng/ml IL-1ß for 24 hr or 48 hr. A portion of each sample was treated with RNase-I to generate ribosome footprints used for Ribo-seq analysis, while an undigested portion was used for RNA-seq.