Reagent	Volume (μl) per reaction	Final concentration (vol = 20 μl)
Water	2 μl
T4 RNA ligase reaction buffer, 10×	0.5 μl
10 mM ATP	0.5 μl	1 mM
Barcoded 5′ adapter	1 μl	5 μM
RNA ligase 1	1 μl	0.5 U μl−1
Mix total volume	5 μl