RG2-coverage.bw

30 x 10^6 Panc-1 cells, treated or not for 16 h with 1 µM PDS, were collected and then lysed for 10 min. with Hypotonic Lysis buffer (5 mM Pipes, 85 mM KCl, 0.5 % NP40) containing protease inhibitor cocktail. Pellets containing nuclei were discarded while cytoplasmic fraction was diluted 1:1 in G4RP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT and 0.5 % NP40 in nuclease-free water). After preclearing, lysates were incubated with Incubate the beads with 3 μg of BG4 (Merck) antibody and 8 μl Dynabeads Protein G for 6h at 4°C. After washings, beads were incabated with 1ml of Tri-Reagent (Sigma-Aldrich, Germany) to extract RNA with a phenol/chloroform extraction method. RNA was DNAseI (Thermo Fisher Scientific) treated to digest genomic DNA. Concentration, integrity and purity of RNA were checked using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit).