GSM6788335: RS4.11 DMSO 24h GRO-seq biol rep2; 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 of PBST wash buffer four times (refer to detailed protocol in Roberts et al. 2015). The cDNA template was PCR amplified (Illumina barcoding) for 12 cycles and size selected to 225–350 bp length. The ready libraries were sequenced with Illumina Hi-Seq2000 (GeneCore, EMBL Heidelberg, Germany).