Rna polii n 20

Epidermal cells were grown to 60–70% confluency and transfected with control or RN7SK siRNA 5 (8 × 15 cm dishes per siRNA). 18 h after transfection cells were fixed with 1% formaldehyde for 10 min at room temperature. Cross-linking was terminated with 2.5 M glycine for 5 min. Cell pellets were recovered by centrifugation at 1350 × g for 5 min at 4 °C. Cell pellets were recovered and chromatin was isolated and sonicated for 17 cycles of 30 s with an output of 30 W, using an automated sonicator (3000; Misonix)^{68 (link)}. Immunoprecipitation was carried out overnight at 4 °C using 5 μg of H3K4Me1 (Abcam) or 10 μg of H3K27Ac (Abcam) or RNA Pol II N20 (Santa Cruz) antibodies previously bound to Dynabeads protein G (Thermo Fisher Scientific). DNA-protein complexes were eluted in 200 μl of elution buffer (50 mM Tris pH 8, 1 mM EDTA, 1% SDS) by incubating at 65 °C with brief agitation every 2–3 min. Cross-links were reverted both in the immunoprecipitated samples and the whole-cell extract, separated from the sonicated material before immunoprecipitation, by incubating overnight at 65 °C. DNA was then purified by phenol-chloroform extraction. Libraries were prepared using NEXTflex Rapid DNA-Seq Kit (Bioo Scientific). Four replicates per sample were sequenced on an Illumina HiSeq 2500 platform. Each ChIP-seq experiment was performed once with four technical replicates per sample.