AGO2-bound RNA Immunoprecipitation

Co-immunoprecipitation of AGO2-bound RNAs was conducted as reported earlier [21 (link)]. In brief, the 293T cells were first cross-linked with 0.3% formaldehyde. Approximately 1.0 × 10⁷ cells were collected and lysed. Then, the protein A/G magnetic beads (Cell Signaling Technology) coupled with AGO2 (Cell Signaling Technology) or IgG control antibody were added into the cell lysates, and incubated at 4°C overnight. After extensive washing, AGO2-bound RNAs were eluted using elution buffer containing proteinase K, followed by reverse cross-linking. The eluted RNA was purified and reverse transcribed to cDNA according to the manuals.