Bromouridine (BrU)-labeled RNA Immunoprecipitation and Bru-Seq

BrU labeling of newly synthesized RNA and BrU-RNA isolation were performed following the method established by Ljungman’s group²². Briefly, MCF-7 cells transfected with either control siRNA or lincNORS siRNA were cultured in hypoxic conditions. BrU (Sigma-Aldrich 850187) at a final concentration of 2 mM was added to the plate when cells reached ~80% confluency. After labeling for 30 min, cells were harvested for total RNA extraction. BrU-labeled RNA was immunoprecipitated from the total RNA using anti-BrdU antibody (BD Pharmingen, 555627) conjugated with Dynabeads® Goat anti-mouse IgG beads (Invitrogen #11033). After 1-h incubation, beads were washed three times with 0.1% BSA in DEPC-PBS to remove unbound RNA then heated at 95 °C for 10 min to recover the BrU-RNA from the beads. The BrU-RNA and the input RNA were analyzed using qPCR. The same BrU-RNA isolation procedure was performed with MCF-7 cells cultured in 21% or 1% O₂ and the BrU-labeled RNA was sequenced at the University of Michigan Sequencing Core using Illumina HiSeq2000 sequencer.