Reverse Transcription and RT-qPCR Protocol

Total (input) or AGO-immunoprecipitated RNA was reverse transcribed^{82 (link)}. Briefly, 100–500 ng of input or IP RNA was reverse transcribed in a final volume of 10 µL containing 2 µL 5× ProtoScript II reaction buffer (NEB), 25 µM ATP, 25 µM dNTPs, 50 µM RT primer (IK-44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of protoscript II reverse transcriptase (NEB). Reactions were incubated at 42 °C for 1 h followed by enzyme inactivation at 95 °C for 5 min. Real-time quantitative reverse transcriptase PCR (RT qPCR) was performed using a LightCycler 480 II (Roche) with SensiFAST SYBER No-ROX (Bioline Meridian Biosystems) using the gene-specific primers listed in Supplementary Table 2. PCR was carried out in technical triplicates using the following cycling conditions: 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. A melting curve was generated at the end of the amplification in every run to confirm primer specificity. Threshold cycle (C_t) values were determined by calculating the second derivative maximum of three technical triplicates for each sample. Data were analysed using Prism-GraphPad Software v8.4.0.