GSM8403714
After 2 hours of BrU labeling, the culture medium was removed, and cells were harvested with Trizol LS for RNA extraction (Invitrogen #10296010). Purified total RNA was treated with TurboDNase (Ambion AM18907) and fragmented by Fragmentation Buffer (Ambion AM8740). BrU-incorporated RNA fragments were immunoprecipitated with mouse monoclonal anti-BrdU antibody 3D4 (BD Biosciences 555627 Lot 7033666). Immunoprecipitated RNAs were used to construct DNA sequencing libraries using NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs E7760).