2.4. RNA Immunoprecipitation (RIP)

Magna RIP Kit (Millipore, Darmstadt, Germany) was used according to the manufacturer’s protocol. For each RIP reaction, 100 μL of cellular pellet from HEK-293 cells was fixed with 1% formaldehyde in PBS at room temperature for 10 min. Cross-linking reaction was stopped by adding 590 μL of 2.5 M glycine. Fixed cells were subsequently harvested and resuspended in RIP lysis buffer supplemented with protease/RNAse inhibitors. Lysate was obtained using a dounce homogenizer on ice (dounced 10 times for releasing nuclei) followed by incubation on ice for 15 min. An equal volume of RIP lysis buffer was added to the cellular pellet. From the solution, 10 μL (10%) of lysate was removed and stored as an “input”. For each RIP reaction, 100 μL of lysate was mixed with 5 μg of rabbit anti-IgG (immunoglobulin G; negative control provided with the kit), anti-HuR (Millipore, #03-102), and anti-AGO2 (Argonaute 2; Millipore, #03-110) antibodies previously conjugated with protein A/G magnetic beads (provided with the kit). After incubation at +4 °C overnight, RNA-protein immune complex was extensively washed with RIP Wash Buffer (provided with the kit). The cross-linking was reversed by incubation with proteinase K. The immune-precipitated RNA was purified through phenol/chloroform/isoamyl alcohol (5:1:1). The purified immuno-precipitated RNA was treated with DNase I and reverse transcribed using SuperScript VILO Master Mix.