Mapping RNA 3' Ends by RACE

Mapping was performed according to the 3′-RNA ligase-mediated RACE method^{31 (link)} with minor modifications: Total RNA preparations were first 3′-dephosphorylated using T4 PNK for 1 h at 37 °C without ATP and pre-adenylated linker (Universal miRNA cloning linker, NEB) ligation was performed during 4 h at 22 °C in the presence of truncated RNA ligase 2 (NEB)^{30 (link)}. Reverse transcriptase reactions were performed using primer prE complementary to the linker sequence. PCR primer prF specific to mRNA1, or primer prK specific to (CGA)₄-mRNA, were used with primer prE in PCR reactions (Supplementary Figs. 3a and 6c). PCR products were purified, cloned into Zero Blunt TOPO PCR Cloning vector (Invitrogen), transformed and plasmids sequenced.