Thermo scientific t4 polynucleotide kinase

RNA was extracted from 5 mL cultures prepared as detailed above. Cultures were centrifuged for 10 min at 3,700 g, and the pellet was thoroughly resuspended in 1 mL TRI Reagent® (Sigma-Aldrich). Total RNA was then extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research) with the recommended DNase I treatment, according to manufacturer's instructions. Cappable-seq was carried out as described elsewhere (41) , with the following modifications. Approximately 10 μg of clean RNA was used for each assay. The very first RNA clean-up step was performed using Agencourt® RNAClean® XP Beads (Beckman) to ensure maximum elimination of unincorporated DTB-GTP. The removal of 3' phosphates from fragmented RNA was performed using the Thermo Scientific™ T4 Polynucleotide Kinase (Thermo Fisher Scientific) and its supplied ATP-free buffer. For RNA-seq samples, 800 ng of total RNA was fragmented in 5X RNA Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA) by incubating at 95°C for 7 minutes and quenching immediately on ice. RNA was then purified using the RNA Clean & Concentrator-5 Kit (Zymo Research), according to manufacturer's instructions. The quality and concentration of RNA before and after fragmentation were evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies). Replicates are detailed in Supplementary Table S2.