GSM7174050: PC346C, AR, -R1881; Homo sapiens; ChIP-Seq
PC346C cells were fixed sequentially with di(N-succimidyl) glutarate and 1% formaldehyde to cross-link chromatin. Chromatin was prepared for sonication with 20 mM Tris-HCl pH8, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100. We used 15 cycles of 30 seconds ON, 30 seconds OFF on a Bioruptor Pico sonication device (Diagenode Cat# B01060001) to shear chromatin to 150-200bp fragments. The resulting chromatin extract was incubated overnight at 4°C with 100ul of Dynal Protein G magnetic beads that had been pre-incubated with approximately 10 μg of the appropriate antibody. Beads were washed 1X with the sonication buffer, 1X with 20 mM Tris-HCl pH8, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%Triton X-100, 1X with 10 mM Tris-HCl pH8, 250 mM LiCl, 2 mM EDTA, 1% NP40 and 1X with TE containing 50 mM NaCl. Bound complexes were eluted from the beads (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA and 1% SDS) by heating at 65°C for 1 hr with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Finally ,DNA was precipitated using phenol:chloroform:isoamyl alcohol (PCI).  Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex ChIP-seq kit from BioO Scientific. Libraries were prepared according to illumina's instructions accompanying the DNA sample kit.