GSM7716909: NGN2_AroLITE_12h_r3 TT-Slamseq; Homo sapiens; OTHER
Total RNA was extracted with Trizol (Ambion) and 24:1 chloroform:isoamylalcohol (Sigma) while using 0.1 mM dithiothreitol (DTT) in isopropanol precipitation and ethanol washes. For each sample, 50 µg of total RNA was fragmented with Magnesium RNA Fragmentation Module (NEB), and fragmentation buffer was removed from samples with ethanol precipitation in presence of 0.1 mM DTT. RNA was then resuspended in 350 µl RNase-free water, diluted in biotinylation buffer (200 mM HEPES pH 7.5, and 10 mM EDTA) and topped up with 5 µg MTS-Biotin (previously diluted to 50 µg ml–1 in dimethylformamide) to reach a final volume of 500 µl. The biotinylation reaction was incubated for 30 min at room temperature while keeping samples in rotation and protected from light. Unbound biotin was removed with acid-phenol:chloroform extraction (125:24:1, Ambion) and isopropanol precipitation. Biotinylated RNA was resuspended in 100 µl RNase-free water, denatured in 65 °C for 10 min and then cooled on ice for 5 min. The biotinylated RNA was captured with 100 µl µMACS streptavidin beads (Miltenyi) by incubating for 15 min in rotation while keeping samples protected from light. µMACS columns were equilibrated on magnetic stand with nucleic acid equilibration buffer and two times with biotinylation buffer (20 mM HEPES, 1 mM EDTA, pH 8). Beads were transferred to columns and washed three times with wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl and 0.1 % Tween 20), and labeled RNA was eluted twice with a total 200 µl of 100 mM DTT. RNA was cleaned up with RNeasy Minelute columns (Qiagen) and eluted to RNase-free water with 1 mM DTT. 4sU residues of RNA were alkylated with iodoacetamide treatment (10 mM iodoacetamide in 50 mM NaPO4, pH 8 and 50 % DMSO) by incubating samples in 50 °C for 15 min, followed by quenching with 20 mM DTT. RNA samples were purified with ethanol precipitation and treated with Turbo DNase (Invitrogen). Sequencing libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos (NEB), according to manufacturer's instructions, except using 8 min incubation time in fragmentation ste