title: GSM8627570: K562,EC100; MED23-FKBP,2,DMSO; Homo sapiens; OTHER
library_construction_protocol: After indicated treatment of cells, nascent RNA was labeled by addition of BrU (5-Bromouridine, Sigma-Aldrich, #850187) to K562 cell culture medium in 2mM final concentration for 6 minutes. Cells were then immediately put on ice, centrifuged for 4 minutes at 200G at 4 degrees and resuspended in TRIzol (Invitrogen). RNA was isolated using conventional TRIzol extraction and isopropanol precipitation, and final pellet was resuspended in DEPC. 50ug of total RNA was used for BrU pulldown, following a protocol adapted from Roberts 2015. In short, per sample 6ug of Anti-BrdU antibody (BD Pharmingen, #555627) was incubated with 30ul of Protein G Dynabeads (ThermoFisher) for 10 minutes at room temperature, and washed in PBS-T. Beads were then blocked with 0.05 ug/ul BSA in PBS-T for 30 minutes and washed with PBS-TR (PBS-T supplemented with RNAseOUT (ThermoFisher)). RNA was diluted in PBS-TR and heated at 65 degrees for 5 minutes, after which it was added to antibody-coated beads at room temperature for 1 hour in 300ul total volume. Beads were then washed three times in PBS-TR, and BrU-labeled enriched RNA was extracted using TRIzol. Pellets were resuspended in 5ul DEPC, which was used as input for NEBNext Ultra II Directional RNA Library Prep Kit (NEB, cat. no. E7645).