GSM4890972: IgG ChIP MED19-LNCaP CS R1881 2; Homo sapiens; ChIP-Seq
Cell pellets were resuspended in nuclei isolation buffer (50 mM Tris–pH 8.0, 60 mM KCl, 0.5% NP40), nuclei collected, and resuspended in sonication buffer (RIPA buffer).  Samples were sonicated in TPX PMP tubes (Diagenode) for 60 min (30 sec. on, 30 sec. off) in a Bioruptor sonicator (Diagenode). Inputs (10%) were collected and supernatants were then incubated overnight with the following antibodies pre-incubated with Protein A and Protein G Dynabeads (Invitrogen): a mixture of AR antibodies (AR 441 mouse monoclonal, Santa Cruz Biotechnology, cat # sc-7305) and (AR D6F11 rabbit monoclonal, Cell Signaling Technology, cat # 5153); DYKDDDDK Tag (FLAG epitope) (Cell Signaling Technology, cat # 14793); or H3K27 acetylation (Active Motif, cat # 39034).  Control ChIPs were performed with normal mouse IgG (Santa Cruz Biotechnology, cat # sc-2025) and normal rabbit IgG (Sigma Aldrich, cat # 12-370). Immunocomplexes were then washed and cross-linking reversed overnight at 65 °C with 5 M NaCl. DNA was isolated with the Zymo Chip DNA Clean and Concentrator kit (Zymo Research). libraries were prepared according to the protocol described in PMID: 30679424.  Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from a 10% polyacrylamide gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Sequencing was performed using Illumina HiSeq4000 Sequencing (HiSeq 4000 Single Read 50 Cycle Lane).