RNase treatment followed by co-IP

On tagged proteins: Cells were harvested, washed twice with ice-cold 1× PBS (Gibco, Life Technologies), and resuspended in 550 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl₂, 0.1% NP-40), supplemented with Complete-EDTA-free Protease Inhibitor Cocktail (complete Mini; Sigma Aldrich). Cells were lysed by 30 min incubation on ice and debris were removed by 15 min centrifugation at 2000 g and 4°C. Lysate was treated or not with RNase A/T1 mix (Thermo Fisher Scientific) and place at 37°C 30 min. An aliquot of the cleared lysates (25 μL) was kept aside as protein Input and another aliquot (25 μL) was kept to assess RNase treatment efficiency. Co-IP was led as previously described.

Total RNA was extracted using Tri-Reagent Solution (Fisher Scientific; MRC, Inc) according to the manufacturer’s instructions. RNA integrity upon treatment was verified on an 1% agarose gel containing ethidium bromide 10 mg/mL (Invitrogen, Thermo Fisher Scientific) and revealed under UV on Gel DocEZ system (Bio-Rad).