Protein g plus agarose beads

ChIP experiments were performed as described previously (http://younglab.wi.mit.edu/hESRegulation/Young_Protocol.doc). In brief, chromatin was cross-linked for 10 min at room temperature with 11% formaldehyde/PBS solution added to cell culture medium. Cross-linked chromatin was then sonicated, diluted and immunoprecipitated with Protein G-plus Agarose beads (Bio-Rad®) prebound with antibody (anti-AR, N-20, SC-816; anti-AR, C-19, SC-815 from Santa Cruz; anti-H3K4me1, ab8895; anti-H3K4me2, ab7766; anti-H3K27Ac, ab4729; anti-H2A.Z, ab4174; Pk-tag antibody (V5-tag antibody), ab9116 from Abcam) at 4°C overnight. Precipitated protein-DNA complexes were eluted and cross-linking was reversed at 65°C for 16 h. DNA fragments were purified and analyzed by real-time PCR. ChIP-seq libraries were prepared using previously described methods (23 (link)). High-throughput sequencing (51 nt, pair-end) was performed using the Illumina HiSeq™2000 platforms at the Mayo Genome Core Facility. Real-time quantitative PCRs were carried out in a Bio-Rad CFX96™ Real-Time System, using SYBR green PCR master mix (Bio-Rad, Hercules, CA, USA). Reactions were carried out in triplicate and with biological replicates. Primers are shown in Supplementary Table S10. ChIP-qPCR data were analyzed as % input after normalizing each ChIP DNA fraction's Ct value to the Input DNA fraction's Ct value.

ChIP experiments were performed as described previously (56 (link)). In brief, chromatin was cross-linked for 10 min at room temperature with 11% formaldehyde/PBS solution added to the cell-culture medium. Cross-linked chromatin was then sonicated, diluted, and immunoprecipitated with Protein G Plus agarose beads (Bio-Rad) prebound with antibody at 4 °C overnight. Precipitated protein–DNA complexes were eluted, and cross-linking was reversed at 65 °C for 16 h. DNA fragments were purified and analyzed by qPCR. ChIP-qPCR data were analyzed as % input after normalizing each ChIP DNA fraction’s Ct value to the input DNA fraction’s Ct value.