T4 rna ligase 2 truncated kq

RNA library preparation was adapted from [44 (link)] and performed as described [22 (link), 38 (link)]. Briefly, RNAs were fragmented with the addition of 20 mM MgCl₂ at 94 °C for 10 min. Fragments were separated on a denaturing 15% gel, and fragments between 50–80 nt were cut out and RNA was eluted overnight in RNA gel extraction buffer (0.3 M NaOAc, pH = 5.5, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.25% vol/vol SDS (sodium dodecyl sulfate). Eluted RNA fragments were dephosphorylated using T4 PNK (NEB), ligated to an adaptor using T4 RNA ligase II truncated KQ(NEB), and then gel purified. TGIRT III (InGex) was used to reverse transcribe RNAs in 50 mM Tris HCl, pH = 8.3, 75 mM KCl, 3 mM MgCl₂, 1 mM dNTPs, 5 mM DTT, and 10 U SUPERase·In (Invitrogen) for 1.5 h at 60 °C. The RNA was later hydrolyzed by 250 mM NaOH and the cDNA was circularized using CircLigase II (Lucigen). The circularized cDNA was shipped to the Scripps La Jolla Genomics Core for final library preparation with Illumina sequencing adaptors and sequenced using single-end sequencing on the Illumina NextSeq 500 platform.

A dephosphorylation step is necessary to remove 3’ end phosphates that prevent adapter ligation. Beads were resuspended in 20 µL of dephosphorylation reaction [4 µL of 5x PNK reaction buffer, 0.5 µL T4 PNK (NEB M0201), 0.5 µL RNasin (Promega N2111), 15 µL H₂O] at 37° C for 20 min followed by washes with PNK buffer. Beads were resuspended in in 20 µL ligation mixture [2 µL of 10x RNA ligation buffer, 2 µL of T4 RNA ligase II truncated KQ (NEB M0373), 0.5 µL of SUPERaseIN, 1.5 µL of 20 µM pre-adenylated L3-App adapter, 4 µL of PEG8000, 10 µL H₂O] at 16° C for overnight.