GSM6788332: RS4.11 AZD1775 10h GRO-seq biol rep1; Homo sapiens; OTHER
Five million nuclei were collected from RS4;11 cells after 4, 10 and 24 h of 1 mircomolar AZD1775(Wee1i) or DMSO (vehicle) treatment. The nuclear run-on reaction buffer (496 mM KCl, 16.5 mM Tris-HCl, 8.25 mM MgCl2 and 1.65% Sarkosyl (Sigma-Aldrich, Steinheim, Germany) was supplemented with 1.5 mM DTT, 750 uM ATP, 750 uM GTP, 4.5 uM CTP, 750 uM Br-UTP (B7166,Sigma-Aldrich) and RNAse inhibitors (RNase Inhibitor (Thermo Fisher, Carlsbad, CA, USA, and RNasin Plus RNase Inhibitor Promega). To each 100 ul of nuclei samples 50 μl of the run-on reaction buffer was added and incubated for 5 min at 30C. RNA was collected using Trizol LS, fragmented and run-on reaction products were immuno-purified two times using anti-Br-UTP antibody (ab-6326, Abcam) bound to 30 μl of magnetic beads (Protein G DynabeadsThermo Fisher Scientific) and washed with 300 μl 