design: Prior to harvest, LNCaP cells were cultured in charcoal-stripped FBS supplemented media for at least 48 hours and subjected to 5 hours of ethanol (vehicle) or 1nM R1881. Briefly, cells were crosslinked in 1% formalin for 10 min and quenched with 1.25M Tris-Cl pH 8. After two washes in ice cold 1XPBS, nuclei are extracted with ChIP lysis buffer (10mM Tris-Cl pH 8, 10mM NaCl, 1mM EDTA, 1mM EGTA, 0.2% Nonidet P-40, 1mM DTT, protease inhibitor cocktail). Chromatin is fragmented by MNase digestion (MNase digestion buffer contains 20mM Tris-Cl pH 7.5, 15mM NaCl, 60mM KCl, 1mM CaCl2), the reaction is quenched with final concentration of 20mM EDTA and the nuclei are fully broken by sonication in SDS lysis buffer (1%SDS, 10mM EDTA, 50mM Tris-Cl pH 8). Sonicated samples were then quenched with of 2XStop/ChIP buffer (100mM Tris-Cl pH 8, 20mM EDTA, 200mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate) and centrifuged at max speed at 4 degree for 10 min to remove debris, with 1% reserved as input and the remaining chromatin samples were incubated with Protein A/G magnetic beads (Millipore) and indicated antibodies overnight at 4 degree with gentle rotation. Protein-bound beads were then washed with ice cold Low Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-Cl pH 8, 150mM NaCl), High Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-Cl pH 8, 500mM NaCl), ice cold LiCl buffer (0.25M LiCl, 1% IGEPAL CA630, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-Cl pH 8) and ice cold TE buffer (10mM Tris-Cl pH 8, 1mM EDTA) respectively. DNA is then eluted and de-crosslinked with ChIP elution buffer (50mM Tris-Cl pH 7.5, 50mM NaCl, 5mM EDTA, 1%SDS) at 65 degree overnight, then incubated with proteinase K for at least 2 hours before DNA is purified by column purification. ChIP enrichment is then submitted for library construction and next generation sequencing.