title: GSM5824382: IPA_HP_Silv_n2; Homo sapiens; OTHER
library_construction_protocol: For preparation of polysomal and whole cytosolic fractions, cells were incubated at 37 °C with 100 μg/mL cycloheximide in fresh medium for 5 min. Cells were then washed, scraped into ice-cold PBS supplemented with 100 μg/mL cycloheximide, centrifuged at 400g. for 5 min. The cell pellets were resuspended into 400 μL of LSB buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5 M sucrose, 1 mM DTT, 100 U/mL RNasOUT and 100 μg/mL cylcoheximide). After homogenization, 400 μL LSB buffer supplemented with 0.5 % Triton X-100 and 0.5% sodium deoxycholate was added. Samples were incubated on ice for 30 min and centrifuged at 12,000 g for 15min at 4 °C. The lysates were then loaded onto a 5-50 % sucrose density gradient and centrifuged in an SW41 Ti rotor (Beckman) at 36,000 rpm for 2 h at 4 °C. Fractions were monitored and collected using a gradient fractionation system (Isco). RNA was extracted from the five heaviest polysomal fractions (pooled). RNA from whole or fractionated cell lysates were extracted with TRIzol Reagent (TRIzol-LS for polysomes; ThermoFischer Scientific) according to the manufacturer's instructions, and 1 µl of GlycoBlue (ThermoFisher Scientific) was added for RNA precipitation. RNA was treated with TURBO DNase I (ThermoFisher Scientific) and quantified using a Nanodrop 2000 spectrophotometer (ThermoFischer Scientific). For sequencing, RNA samples were analyzed using an RNA 2100 Bioanalyzer (Agilent). 3'-seq libraries were prepared with QuantSeq 3' mRNA-Seq Library Prep Kit REV for Illumina (Lexogen) using 500 ng (for polysomal or whole cytosolic RNAs) of DNase 1 treated RNA (n=3 for each condition) following manufacturer's instructions. Purified libraries were quantified with Quant-iT Picogreen dsDNA kit (ThermoFisher Scientific) and run on Experion automated electrophoresis system (BIO-RAD). Pooled libraries were quantitated by qPCR (KAPA Library Quantification Kits Illumina Platforms, Roche), diluted to 12 pM, and subjected to single-end, 51 bp sequencing using the NovaSeq 2500 machine (Illumina).