RNase A treatment

RNase A treatment

RNA concentrations from clarified cell lysates were measured using Qubit RNA HS (High Sensitivity) Assay kit (Thermo Q32855) or Qubit RNA BR (Broad-Range) Assay kit (Thermo Q10211) with a Qubit Fluorometer. Lysates were treated with RNase A (Thermo EN0531) using the following condition – 0.5 µg RNase A was added to 15 µg RNA in a 300 µl reaction volume and shaken at 500 rpm (20 min, 25˚C) on a table-top thermo-mixer (Eppendorf); the reaction was quenched by the addition of 200 units SUPERase·In™ RNase inhibitor (Thermo AM2696). RNase A digested lysates were layered on top of 10-35% sucrose gradients and processed as described below.

Sucrose Gradient Fractionation

Stock solutions of 10x gradient buffer (250 mM HEPES pH 7.4, 1M KOAc, 50 mM Mg(OAc)₂) and 60% (w/v) sucrose in water were prepared, filter-sterilized through a 0.22 µm filter, and stored at room temperature. On the day of the experiment, gradients were prepared from two freshly-made sucrose buffers containing 1x gradient buffer supplemented with 1 mM TCEP, 360 µM emetine, 200 units SUPERase·In™ RNase inhibitor (Thermo AM2696), and sucrose to the appropriate concentration (10% and 50% (w/v) sucrose buffers for undigested samples, and 10% and 35% (w/v) sucrose buffers for RNase A-digested samples). To prepare gradients, 6 ml of 10% sucrose buffer was added to a SW41 ultracentrifuge polypropylene tube (Seton Scientific), after which 6 ml of 35% or 50% sucrose buffer was added to the bottom of the tube using a 10 ml syringe and cannula; 10-50% and 10-35% sucrose gradients prepared on a Biocomp Gradient Master. Gradients were stored at 4˚C until use on the same day. Samples were normalized by measuring the RNA concentration using Qubit RNA HS (High Sensitivity) Assay kit (Thermo Q32855) or Qubit RNA BR (Broad-Range) Assay kit (Thermo Q10211) with a Qubit Fluorometer. Equal RNA load (~15-25 µg, depending on the experiment) was layered on top of each sucrose gradient; gradients were ultra-centrifuged in a Beckman SW41 swinging bucket rotor (40,000 rpm; 105 min). Gradients were fractionated and UV (A260) absorbance across sucrose gradients was measured using a top-down Biocomp Piston Gradient Fractionator™ as per manufacturer’s instructions. For SDS-PAGE and immunoblotting, proteins from individual fractions were TCA-precipitated and stored at -20˚C overnight. The following day, TCA-precipitated fractions were centrifuged at 20,000 x g (30 min, 4˚C), the supernatant aspirated, pellets washed (x 3) in 500 µl acetone and centrifuged at 20,000 x g (10 min, 4˚C), aspirating the supernatant after each wash. After the final wash step, pellets were vacuum-dried briefly (~ 2-3 min, 42˚C) in a vacuum evaporator, resuspended in Laemmli buffer, pH neutralized with Tris-HCl pH 8.0, boiled (95˚C, 5 min) and resolved by SDS-PAGE.