Polysome Profiling of Individual mRNAs

Polysome profiling of individual mRNAs was described before (8 (link)), with the exception that total lysates were used. Macrophages were rinsed in ice cold 1× PBS containing 0.1 mg/ml cycloheximide prior to lysis. Total lysates from 10 × 10⁶ cells were separated on a linear 10–50% sucrose gradient by ultracentrifugation for 2 h at 35 000 rpm using a SW40.1 Ti rotor (Beckman-Coulter). After centrifugation 12 fractions (1 ml each) per gradient were collected using a UA-6 UV/VIS (Teledyne/ISCO Inc.) device. The RNA of each fraction was precipitated overnight with isopropanol and 3 M Na-acetate at –20°C and purified using the NucleoSpin RNA kit (Macherey+Nagel) by dissolving the resulting pellet in buffer RA1 from the kit. Purified RNAs were subjected to cDNA synthesis and qRT-PCR as described above.