T4 polynucleotide kinase

1 μg of DMS-treated RNA, either total, or rRNA-depleted using Ribo-Zero rRNA Removal Kit (Illumina), was fragmented in the presence of 10 mM MgCl₂ at 94°C for 8 min. RNA was end-repaired by treatment with 1 U rSAP (NEB) at 37°C for 30 min. After heat-inactivating the enzyme at 65°C for 5 minutes, RNA fragments were phosphorylated by treatment with 10 U T4 Polynucleotide kinase (NEB) in the presence of 1 mM final ATP at 37°C for 1 h. After reaction cleanup on RNA Clean & Concentrator™-5 columns (Zymo Research), 3′ and 5′ adapters were ligated to RNA fragments using the NEBNext^® Small RNA Library Prep Set (NEB). RNA was then heat-denatured at 70°C for 5 min and incubated at room temperature for 5 min. Reverse transcription was carried out in a final volume of 10 μl, in the presence of 1 mM dNTPs, 10 pmol of SR RT primer (NEBNext^® Small RNA Library Prep Set kit), 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U TGIRT™-III Reverse Transcriptase (InGex), by incubating at 50°C for 5 min, followed by 2 h at 57°C. Template RNA was degraded by adding 1 μl of 5 M NaOH and incubating at 95°C for 3 min. After reaction cleanup, cDNA was eluted in 20 μl nuclease-free water, and barcodes were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext^® High-Fidelity 2× PCR Master Mix.