GSM8545852: C17,TACL-OFF,rep1, BrU-seq; Homo sapiens; OTHER
Cultured cells were incubated with 2mM Bromouridine (BrU, Merck) for 10min and subsequently lysed in TRIzol reagent (Thermofisher). RNA was isolated following the manufacture's protocol. Briefly, lysed cells were mixed with chloroform and centrifuged for 15min. The aqueous phase was transferred to a new tube and mixed with isopropanol. After centrifugation, RNA pellet was washed once with 70% ethanol and dissolved in DEPC water. To capture BrU-labelled nascent RNA, 6ug anti-BrdU antibodies (BD biosciences) pre-coupled with Dynabeads protein G beads (Thermofisher) were incubated with the total RNA for 1h at room temperature. Beads were then washed 3x with PBS/0.1% Tween-20/RNaseOUT. To purify the beads-bound RNA, TRIzol reagent was directly added to the beads and RNA was purified as described above. NGS sequencing libraries were generated using NEBnext Ultra II directional RNA library prep kit (NEB) following manufacture's protocol. Briefly, RNA was fragmented to about 200bp in size. First strand and second strand cDNA were synthesized. Double strand cDNA was then end repaired, poly-A tailed, ligated to NEBnext adapters, and digested with USER enzyme. Annealed libraries were then purified with AMPure XP beads (Beckman Coulter) and PCR amplified with indexing primers for 7 cycles.