Nascent EU-RNA labeling and library preparation

Nascent EU RNA-seq (neuRNA-seq) labeling and capture were done by using Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Kc cells were incubated with 0.2 mM EU for 1 h and RNA was extracted with Trizol (Thermo Fisher Scientific). Next, RNA was chemically fragmented for 5 min at 70 °C with RNA Fragmentation Reagents (Thermo Fisher Scientific), followed by DNase I treatment (Roche). Then RNA was ethanol precipitated after Phenol:Chloroform (Thermo Fisher Scientific) purification. The Click-iT reaction was performed with 0.5 mM biotin azide using 5 µg of EU-RNA, and biotinylated RNA was captured with 12 μL T1 beads. The nascent EU-RNA was used to generate RNA-seq libraries with Ovation RNA-seq Systems 1–16 for Model Organisms (Nugen). Samples were sequenced on HiSeq2500 (Illumina) using 50 bp single-end sequencing at the NIDDK Genomics Core Facility.