The reaction mixture was purified by centrifugation.
After two rounds of 30 min incubation at 37 °C, the reaction mixture was purified with the RNA Clean and Concentrator-5 kit (ZymoResearch, Irvine, CA) following the procedure described for retention of >17 nt RNA fragments.
The obtained mixture was purified by centrifugation.
The crude mixture was purified by C18 SPE.
The crude reaction mixture was purified by column chromatography.
Finally, the mixture was purified by dialysis for 1 week to remove the remaining metal species.
The mixture was purified by centrifugation twice (10,000 rpm for 10 min, at 4°C, hereinafter the same below).
After incubation, the mixture was purified with an ultracentrifugal filter (Amicon Ultra) to remove the side-products.
The reaction mixture was purified using a PD-10 cartridge and QC was performed using a Zenix-C 80Å column.
The radiolabelled mixture was purified through a Sephadex G50 desalting column equilibrated with 0.9 % NaCl containing 0.05 % human serum albumin.
The crude mixture was purified by HPLC, yielding the pure product with a radiochemical yield of 72±8%8 % (n = 12) and a radiochemical purity >95%%.
