RNA 5′ phosphorylation and RNA-5 blocking oligo ligation

To reduce the signal derived from RNA fragmentation during the CMC reaction, ±CMC-treated RNA in 6.5 µL H₂O was mixed with 0.5 µL RNase inhibitor (NEB, M0307L), 1 µL of 10× T4 PNK reaction buffer A, 1 µL of 1 mM ATP, 1 µL of T4 Polynucleotide kinase (PNK), and then incubated at 37°C for 30 min. To ligate the RNA-5 oligo (/5AmMC6/rArCrCrCrA; Integrated DNA Technologies), 1 µL of 10× T4 RNA Ligase Reaction Buffer, 1 µL of 100 µM RNA-5 oligo, 1 µL of 1 mM ATP, 1 µL of RNase inhibitor, 3 µL of DMSO, 2 µL of H₂O, and 1 µL of T4 RNA ligase I (NEB, M0437M) were added to this mixture and incubated at 16°C for 16 h. The reaction was terminated upon the addition of 1.2 µL of 200 mM EDTA.