Rnase cocktail

Digestion and depletion of RNA and/or DNA from input samples and RNP fractions were necessary prior to MS-based proteomic analysis (Supplementary Figs. 5b and 9a–c). For SILAC LC–MS/MS experiments, RNP suspensions were normalized to 3 µg/µL protein-bound RNA in 1% LiDS TE, and 33 µL were used per 100 µL reaction containing 13.3 µL RNase Cocktail, 1× RNase digest buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, and 1 mM EDTA pH 8.0), and 1× protease inhibitors (11836153001, Roche). For the comparative LEAP-RBP experiment, 10 µL of input samples containing 2.0 µg protein/µL were used per 25 µL reaction containing 0.6 µL RNase Cocktail, 1× RNase digest buffer, and 1× protease inhibitors; 20 µL of clRNP fractions containing 0.2 µg RNA-bound protein/µL were used per 50 µL reaction containing 3.1 µL RNase Cocktail, 1× RNase digest buffer, and 1× protease inhibitors. RNase digests were incubated for 2 h at 37 °C and then precipitated with 95% methanol v/v as described above. Each input sample was suspended in TE buffer and DNA was digested using 2.5 µL Turbo DNase (Thermo, AM2238) and a final concentration of 1× Turbo DNase buffer in a 50 µL reaction (15 min, 37 °C) and precipitated with 95% methanol v/v as described above. Pellets were air-dried and submitted for proteomics analysis. For long-term storage, we recommend storing precipitates in 95% methanol at −80 °C.

RNase digestions were performed in separate 0.2 mL thermocycler tubes (10–12 µL reactions) using a maximum of 5 µL of 1% LiDS TE sample suspensions containing <4.0 µg RNA/µL. RNase Cocktail (AM2286, Invitrogen), 10× RNase digest buffer (100 mM Tris-HCl pH 7.5, 1 M NaCl, and 10 mM EDTA), and 25× protease inhibitors (11836153001, Roche) were added at the same time to a final concentration of 2 µL RNase Cocktail/15 µg RNA (Supplementary Fig. 12a), 1× RNase digestion buffer, and 1× protease inhibitors. A minimum of 0.2 µL RNase Cocktail were added regardless of RNA concentration. Samples were mixed by brief vortexing followed by a brief spin in a mini centrifuge (Supplementary Note 2a). Untreated control samples were prepared without RNase Cocktail, and both were incubated for 2 h at 37 °C in a thermocycler with a heated lid (98 °C) unless indicated otherwise in the provided Source Data (e.g., Supplementary Fig. 6b, c). Input samples suspended in 1% LiDS TE were set up as untreated control reactions for SDS-PAGE and were not incubated at 37 °C unless indicated otherwise in the provided Source Data (e.g., Fig. 7i, j and Supplementary Fig. 6b).