Purification of rRNA from Ribonucleoprotein (RNP) Complexes
Ribosomes and ribosomal subunits were collected from sucrose gradients as peak fractions. The sucrose fractions were collected into 15 mL falcon tubes and diluted at least two-fold with the gradient buffer (25mM Tris-HCl pH 7.9, 100mM KCl, 10mM MgCl2). Next, 2.5 vol. of 96% ethanol was added to the samples and incubated at -20°C overnight. The fractions were pelleted via centrifugation for 45 minutes at 4000 rpm +4°C. The pellet was washed with 70% EtOH, and centrifugation was re-applied for 10 minutes. The ribonucleoprotein complexes were suspended in 0.1 mL of MilliQ water, and samples were stored at -20°C.
The rRNA was purified with phenol-chloroform extraction. The samples were kept on ice, and 1% SDS-containing phenol was added to the samples. Samples were vortexed vigorously for 10 s, kept on ice for 5 min, and centrifuged at 16,200xg at +4°C. The water phase was transferred to a new microfuge tube into which chloroform:phenol mixture (1:1) was added and vortexed for 10 seconds. This step was repeated, using only chloroform to avoid phenol carryover. The water phase was transferred to a new microfuge tube, and the RNA was precipitated with 2.5 vol. ethanol at -20°C for 1 hour. The pellet was washed with 70% EtOH and dried at room temperature for 5 minutes. The purified RNA was dissolved in ultra-pure distilled water.