GSM7730823: ST2 DEXA 48h GRO-seq biol rep4; Mus musculus; OTHER
Five million nuclei were collected from ST2 cells after DMSO (vehicle) treatment. The nuclear run-on reaction buffer (496 mMKCl, 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 uMATP, 750 uMGTP, 4.5 uMCTP, 750 uMBr-UTP (B7166,Sigma-Aldrich) andRNAse inhibitors (RNase Inhibitor (Thermo Fisher, Carlsbad, CA, USA, andRNasin Plus RNase Inhibitor Promega). To each 100 ulof nuclei samples 50 μlof 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 GDynabeadsThermo Fisher Scientific) and washed with 300μlof 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).