GSM8827969: MEF_PUS10KOinp_n1; Mus musculus; RNA-Seq
Cells were incubated for 10 min at 37oC in media supplemented with 10 mg/mL cycloheximide (CHX) (Sigma-Aldrich). After washing with ice-cold PBS containing 10 mg/mL CHX cells were lysed in 450 ml polysome lysis buffer (PLB) with 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 0.25% NP-40, 0.1% Triton X-100, 320 U/mL SUPERase-In RNase Inhibitor (Thermo Fisher Scientific), 150 mg/mL CHX (Sigma-Aldrich) and 20 mM DTT. Lysates were incubated on ice for 40 min, mixed every 10 min and cleared at 15,000 rpm for 10 min at 4oC. Equal amounts of lysates were layered onto a linear 10%–60% sucrose (Thermo Fisher Scientific) (w/v) gradients in 25 mM Tris-HCl pH 7.4, 25 mM NaCl, 5 mM MgCl2, 0.1 mg/mL heparin and 2 mM DTT in nuclease-free water. Samples were centrifuged using a SW41Ti rotor (Beckman) for 2.5 hr at 37,000 rpm at 4oC and fractioned using a BioComp Gradient Station (BioComp). To each 650 μL fraction, 800 μL of TRIzol was added and samples were immediately snap-frozen on dry ice. For individual sucrose gradient fractions, RNA isolation was carried out using Direct-Zol RNA Miniprep (Zymo Research) with the in-column DNAse treatment according to manufacturer's protocol. For normalization, 5 ng of an in vitro-transcribed luciferase RNA (5 ng) was added to each sample before RNA isolation. Sequencing libraries were generated using 500 ng of total or polysomal RNA with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to the manufacturers' instructions. Libraries were sequenced on an Illumina NovaSeq 6000 with paired end 150 cycles.