title: GSM1174491: LuCaP_ARplus; Homo sapiens; ChIP-Seq
library_construction_protocol: The chromatin was sonicated to reach a fragment size of 150-300 bp with Bioruptor UCD-200TM-EX instrument (Diagenode Inc., Liège, Belgium), diluted 1:10 in 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl containing 2X protease inhibitor and precleared for 1 hour with 100 μg of yeast tRNA (Invitrogen Inc., Carlsbad, California, USA) and 20 μl of preimmune rabbit serum (Santa Cruz Inc., Santa Cruz, California, USA) followed by 1 hour incubation with 40 μl of 50% slurry of Gammabind Sepharose beads (GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA) at 4°C. The beads were precipitated and 10 μg of normal rabbit IgG (Santa Cruz Inc., Santa Cruz, California, USA) or 10 μg of anti-AR (clone N20; Santa Cruz Inc., Santa Cruz, California, USA) or 10 μl of anti-AR polyclonal antibody (AR3) (provided by one of the authors: O. A. J.) (Karvonen et al., 1997, Thompson et al., 2006) were added to the supernatant for overnight incubation. The complexes were then incubated with 70 μl of Gammabind Sepharose beads (GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA) 50% slurry for 4 hours and precipitated. The beads were washed sequentially for 10 min ach in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, and 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, and 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl). Beads were then washed two times with TE buffer. The complexes were then eluted from the beads incubating for 15 minutes at 37°C twice with 1% SDS, 0.1 M NaHCO3. The eluates were pooled and treated for 1 hour at 37°C with RNAase A (Qiagen Inc., California, USA) and 1 hour with proteinase K (Finnzymes Oy, Espoo, Finland), then heated at 65°C for 6 3 hours to reverse the formaldehyde cross-linking. The DNA fragments were purified with a DNA purification kit (QIAquick PCR purification Kit, Qiagen Inc., California, USA) and eluted in 100μl of elution buffer. The libraries of ChIP DNA were prepared and sequenced with Genome analyzer II (Illumina Inc., San Diego, California, USA) according to the manufacturer’s protocol. Each sample was sequenced in one lane. The raw reads were obtained using Illumina analysis pipeline. Standard illumina protocol one sample per illumina lane