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

We optimized a 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 Riboseq 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 in astrocytes 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. Three biological repetitions containing qual number of primary astrocytes were lysed using the ‘standard’ or ‘optimized’ extraction method followed by pelleting the heavy complexes from each lysate through 1M sucrose cushion. Equal emount of mRNA from each pellet was sequenced by RNA-seq, representing the population of transcripts that was in association with heavy complexes (RNPs) whithin the cells.