GSM8794541: OS-RC-2, Veh, Rep1; Homo sapiens; OTHER
1×107-2×107 adherent cells were washed once with ice-cold PBS and subsequently lysed/scraped directly from the plates on ice using a polysome extraction buffer [(25 mM HEPES pH 7.5, 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 1% Triton X-100, 0.1 mg/ml cycloheximide, 0.05 U/l RNase inhibitor (Invitrogen AM2696), and 1× Roche-complete EDTA-free protease inhibitor cocktail (Roche 4693132001)]. The lysates were homogenized with 5-6 strokes in a glass Dounce homogenizer on ice, then cleared at 10,000xg for 5 min at 4°C. Cleared lysates were loaded on top of 10-50% linear sucrose gradients (mixed using a BioComp gradient mixer gradients containing 25 mM HEPES pH 7.5, 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 0.1 mg/ml cycloheximide, and 1× Roche-complete EDTA-free protease inhibitor cocktail) and centrifuged in a Beckman Coulter ultracentrifuge at 40,000 rpm for 3 h at 4°C in a SW41Ti rotor. After centrifugation, gradients were fractionated and analyzed using a BioComp fractionator monitoring absorbance at 260 nm. Polysome profiles were manually inspected to determine fractions containing polysomes, and these fractions were pooled for RNA purification (polysome-containing fractions were identified as all fractions heavier than the monosome peak which was excluded). RNA was purified from pooled polysome sucrose gradient fractions using Trizol LS (Invitrogen 10296028) followed by sequential isopropanol and ethanol precipitations. RNA quality was analyzed using an Agilent RNA TapeStation (RIN values all > 9). Libraries were prepared using SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara 634873). The sequencing libraries were quantified using Qubit HS DNA assays and an Agilent High Sensitivity TapeStation. The final pool was sequenced with paired-end 150 bp reads on an Illumina NovaSeq 6000 at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.