Illumina HiSeq 2500 paired end sequencing; Genome-wide profiling of H3K27ac, H3K4me1, H3K27me3 and H3K9me3 (ChIP-seq) in U2OS-GR and U2OS-AR cells
Genome-wide profiling of H3K27ac, H3K4me1, H3K27me3 and H3K9me3 (ChIP-seq) in U2OS-GR and U2OS-AR cells
ChIP-seq was performed on 3 million cells per sample. Cells were cross-linked with 1% formaldehyde for 3 min and harvested. Cells where grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS. U2OS-GR cells were treated for 90 minutes with 1 µM dexamethasone or 0.1% ethanol as vehicle control. U2OS-AR cells were treated for 4 hours with 5 nM R1881 or 0.1% DMSO as vehicle control. Cells were resuspended in 600 µl RIPA Plus (TE Buffer with 0.1%SDS; 1:200 Complete protease inhibitor [Roche], 1:200 PMSF) and incubated for 20 minutes on ice. Each 200 µl were then transferred to a 1.5 ml Bioruptur PicoTube and sheared for 30 cycles. Samples were adjusted with 200 µl RIPA Plus Buffer. The IP was performed using the IP-Star automated system (Diagenode). The sample and input fraction were adjusted with 100 µl ChIP elution buffer, 4 µl 5M NaCl were added and incubated at 65 °C overnight. 4 µl RNAseA 5 mg/ml were added and incubated 1 h at 55°C. 3 µl Ptoteinase K were then added and the samples were incubated for 4 h at 55 °C. The DNA was purified with the Zymo Clean and Concentrator Kit. Antibodies used: H3K27ac, C15410196 (Diagenode 1µg /ChIP); H3K4me1, C15410194 (Diagenode 1µg/ChIP); H3K9me3, C15410193 (Diagenode 1µg/ChIP); H3K27me3, C1541095 (Diagenode 1µg/ChIP)  Sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep kit (NEB E7370) according to manufacturer's instructions.