GSM3415515: BRICseq_Nat10_KO2_0hr; Homo sapiens; OTHER
Cells were incubated in complete DMEM medium containing 150 μM BrU (Sigma-Aldrich) for 24 hr. Cells were washed twice with PBS and medium was replaced with complete DMEM medium containing 150 μM Uridine (Sigma-Aldrich) for 0, 2, 4, 8 and 16 hrs. After each time point, medium was removed and Trizol was added directly to culture dishes. Illumina libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina Total RNA was purified from cultured cells using Trizol (ThermoFisher) followed by treatment with TurboTM DNAse (ThermoFisher). Enrichment of polyadenylated RNA (poly(A)-RNA) from DNAse-treated total RNA was carried out by two rounds of selection with Oligo-(dT)25 Dynabeads (ThermoFisher) according to the manufacturer's instructions. Enrichment efficiencies were estimated with Bioanalyzer picoRNA chips (Agilent Technologies, Santa Clara, CA).
GEO
GSM3415515