GSM5159858: TILAC_293t_500nMFP_s4UFP_s6GCntl_Rep2; Homo sapiens; RNA-Seq
RNA was harvested by two different protocols. For normal RNA sequencing, cells were resuspended in Trizol reagent, followed by chloroform extraction and isopropanol precipitation of the RNA. Genomic DNA was depleted using Turbo DNAse, followed by cleanup with RNAclean beads. Total RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018 and Kiefer et al. Journal of the American Chemical Society 2018. To isolate RNA from untreated polysome fractions, each fraction went through 1 phenol:chlorofom isoamyl alcohol extraction and 2 chloroform extractions. RNA was isopropanol precipitated, fractions were combined, and genomic DNA was depleted using Turbo DNase, followed by cleanup with RNAclean beads. Polysome-associated RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018 and Kiefer et al. Journal of the American Chemical Society 2018. To isolate RNA from stressed polysome fractions, each fraction was ethanol precipitated, resuspended and fractions were combined, and then samples were TRIzol extracted.  Genomic DNA was depleted using Turbo DNase, followed by cleanup with RNAclean beads. Total RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018 and Kiefer et al. Journal of the American Chemical Society 2018. RNA sequencing libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).