Fig. 2

AtHEN1 methyltransferase-directed attachment of a sequencing adapter to the 3′ end of small non-coding RNA duplexes. a An overview of AtHEN1 application. b AtHEN1-directed azide-tagging of substrate RNAs with different 3′ terminal nucleotides. 2 μM of methyltransferase and 400 μM Ado-6-azide were incubated with the mixture of four miRNA/miRNA* duplexes (0.05 μM of each) in which particular guide or passenger strand was ³²P-labelled (let-7a*, miR-26a, miR173A or miR-210* with C, U, A or G at the 3′ end, respectively). After the treatment with sodium periodate samples were analysed by denaturing PAGE. c ³²P-miR173A/miR173* is efficiently attached to 2U and 2C alkyne-adapter/RT primer. Click reaction was carried out under the same conditions as described in Fig. 1c. d cDNA synthesis from miR173A coupled with 2U or 2C alkyne-adapter. 10 nM of RNA-DNA conjugate were reverse transcribed using ³²P-RT primer as described in Fig. 1d