GSM4852327: shUNR poly 12dpi, replicate4; Mus musculus; RNA-Seq
Polysome Profiling was adapted from Liang S. et al, 2017. Media was discarded from shCTRL and shCSDE1 cells (12 dpi – one 100 mm plate per condition), plates were placed on ice and immediately washed in an ice-cold solution of 1x phosphate-buffered saline (PBS) and cycloheximide (100 ug/mL, Sigma-Aldrich). Cells were then lysed in 200 ul hypotonic lysis buffer (5 mM Tris–HCl, pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 100 ug/mL cycloheximide, 2 mM DTT, 0.5% Triton, 0.5% sodium deoxycholate) and left on ice for 10 min. Extracts were cleared by centrifugation at 16'000 rcf for 7 min and the OD was measured (between 2.5 and 4 OD per experiments were loaded onto the gradient). Samples were brought to a final volume of 300 ul if necessary with lysis buffer and loaded on a non-linear sucrose gradient prepared by layering different percentage of sucrose solutions (5%, 34% and 55%) into an Open-Top Thinwall Ultra-Clear Tube (Beckman, 344059). A 5X gradient buffer (100 mM HEPES pH 7.6, 500 mM KCl, 25 mM MgCl2) was used to prepare sucrose solutions (1x final concentration of gradient buffer [v/v] adjusted with water). The samples were centrifuged at 36'000 rpm for 2 hrs at 4ºC in a SW 41 Ti rotor and a Beckman Coulter Ultracentrifuge Optima L-90K. Samples were eluted using the gradient station (BioComp) and fractions (1-6 for monosomal RNA and 11 and 12 for polysomal RNA) were collected with Gradient Fractionator (BioComp) coupled with a fraction collector (Gilson). The precise location of the fractions along the UV-tracing was monitored using the gradient profiler v1.25 (BioComp) software. TRIzol reagent (Invitrogen) was immediately added to each fraction and the RNA was extracted according to the protocol of the manufacturer. Poly-A RNA selection protocol