C15410196

ChIP in LNCaP was performed according to published protocols^{36 (link)}. Ten million cells were fixed with 1% formaldehyde at room temperature for 10 min and quenched. Cells were collected in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor (#11873580001, Roche) in PBS)^{37 (link)}. Chromatin was sonicated to 300–800 bp using a Covaris E220 sonicator (140 watt peak incident power, 5% duty cycle, 200 cycleburtst). Antibodies (FOXA1, ab23738, Abcam; H3K27ac, C15410196, Diagenode; ASCL1, ab74065) were incubated with 40 μl of Dynabeads protein A/G (Invitrogen) for at least 6 h before immunoprecipitation of the sonicated chromatin overnight. Chromatin was washed with LiCl wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) six times for 10 min sequentially.

For ChIP 2 μl of each antibody was used per 10⁷ cells. For western blotting individual dilutions are indicated. Anti-H3K79me3 (Diagenode C15410068, lot no. A246-0040 for ChIP-qPCR, ChIP-seq, and western blotting (1/5000)); anti-H3K79me2 (ChIP-seq, Millipore 04-835); anti-H3K79me2 (ChIP-qPCR, Abcam ab3594); anti-H3K27ac (Diagenode C15410196, lot no. A1723-0041D for ChIP-qPCR, ChIP-seq, and western blotting (1/20,000)); anti-H3K4me1 (ChIP-seq, Diagenode pAB-194-050); anti-H3K4me1 (ChIP-qPCR and western blotting (1/100,000), Abcam ab8895); anti-H3K4me3 (ChIP-seq, Diagenode pAB-003-050); anti-H4 (western blotting (1/100,000), Abcam ab7311); anti-ELF1 (Bethyl A301-443A, lot no. 1 for ChIP-qPCR, ChIP-seq, and western blotting (1/10,000)); anti-RUNX2 (ChIP-seq, Cell Signalling 8486, lot no. 1); anti-MYB (ChIP-seq, Abcam ab177510); anti-CTCF (ChIP-seq, Millipore 07-729); anti-BRD4 (ChIP-seq, Bethyl A301-985A).

Cells were crosslinked in 1% formaldehyde for 10 min and quenched by glycine. Cell nuclear fraction was extracted by LB1 buffer (50 mM Hepes–KOH, pH 7.5; 1 mM EDTA; 140 mM NaCl; 10% glycerol; Igepal CA-630; 0.25% Triton X-100) followed by washing in LB2 (10 mM Tris–HCL,pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) and LB3 buffer (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% deoxycholate; 0.5% N-lauroylsarcosine). Chromatin was sheared to 300–500 bp by Diagenode bioruptor sonicator and then incubated with antibody-conjugated protein A and G beads overnight at 4 °C with rotation. The antibodies for ChIP-seq were anti-MYC (Santa Cruz, sc-764) or anti-H3K27ac (Abcam, ab4729). Antibodies for ChIP-qPCR were anti-MYC (Abcam Ab32072), anti-AR (Abcam Ab74272), or anti-H3K27ac (DIAGENODE C15410196). All antibodies were used at 4 μg per sample. After washing in RIPA buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Igepal CA630, 0.5% deoxycholate, protease inhibitors and RNase inhibitor), the beads were eluted in elution buffer (0.1 M NaHCO₃, 1% SDS and Proteinase K) for 8–16 h at 65 °C. ChIPed DNA was purified by phenol–chloroform extraction and then used for ChIP-qPCR or ChIP-seq library preparation.