FIGURE 2.

Strategy to make a small RNA library. RNA is ligated to an adenylylated 3′ linker using the truncated RNA ligase 2 while 5′ ppp-RNA is dephosphorylated with PIR-1 at step 1; the reaction is inactivated at 65°C for 10 min and an RT primer is annealed to the 3′ linker at 65°C for 5 min at step 2; a 5′ linker is ligated with the target RNA using T4 RNA ligase 1, while hDCP2 is added to decap capped RNA at step 3; an RT is performed to obtain the first strand cDNA at step 4; and cDNA is amplified and extended to obtain full-size 5′ and 3′ linkers at step 5.