GSM5065652: LNCaP cells - AR-pS81_rep1; Homo sapiens; ChIP-Seq
For crosslinking: the cells in media were fixed by adding formaldehyde (1%, final concentration) and incubation at room temperature for 10min, followed by quenching with glycine at a final concentration of 0.2M. Wash cells twice with pre-chilled PBS, and then harvest in PBS. The nuclear fraction is extracted by first re-suspending the pellet in 10ml of LB1 buffer (50mM Hepes-KOH, pH7.5; 140mM NaCl; 1mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100) for 10min at 4°C . Cells were pelleted, re-suspended in 10ml of LB2 buffer (10mM Tris-HCl, pH8.0; 200mM NaCl; 1mM EDTA; 0.5mM EGTA) and mixed at 4°C  for 5min. Cells were then pelleted and re-suspended in 300µl of LB3 buffer (10mM Tris-HCl, pH8.0; 100mM NaCl; 1mM EDTA; 0.5mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine) and sonicated with a waterbath bioruptor (Diagenode). Then 30µl of 10% Triton-X100 was added and the lysate was centrifuged for 10min at 20,000 rcf to separate debris. The supernatant was then subjected to pre-clearing and ChIP analysis. ChIPseq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on a NovaSeq targeting 30 million 50bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute.