SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

Blank, Maximilian F.; Chen, Sifan; Poetz, Fabian; Schnölzer, Martina; Voit, Renate; Grummt, Ingrid

ChIP assays were performed as described (1, 12) . Briefly, nuclei were fixed with 1% formaldehyde (10 min, RT), quenched with 0.125 M glycine and lysed in 50 mM Tris-HCl [pH 8.1], 10 mM EDTA and 1% SDS. Chromatin was sonicated to an average fragment length of 200-500 bp, diluted with 5 volumes of IP-buffer (16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100), pre-cleared on protein A/G Sepharose in the presence of 20 mg/ml sonicated Escherichia coli DNA, and incubated with 1-5 g antibodies overnight at 4 • C. Protein-DNA complexes were captured on protein A/G-Sepharose for 1 h, washed twice with low salt buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 5 mM MgCl 2 , 1% Triton X-100), followed by washes with high salt buffer containing 500 mM NaCl, with LiCl buffer (250 mM LiCl, 10 mM Tris-HCl [pH 8.0], 5 mM EDTA, 0.5% Na-deoxycholate, 0.5% Triton X-100) and with TE buffer. After elution, reversal of the cross-links (65 • C, 6 h) and digestion with proteinase K, DNA was purified and quantified by qPCR using gene-specific primers. The ratio of DNA in the immunoprecipitates (upon subtraction of the IgG background) versus DNA in the input chromatin was calculated and normalized to control reactions from mock-transfected cells.