GSM8177991: LNCaP, R1881, 0.1 nM, 24 hours, Input, Rep2; Homo sapiens; ChIP-Seq
Cells were fixed using a final concentration of 1% methanol-free formaldehyde in PBS at for 10 min and quenched with 125mM glycine for 5 min. Cells were then washed twice in PBS Wash Buffer (Active Motif #53046), pelleted, and stored at -80˚C. For sonication, cells were thawed and resuspended in ice cold Buffer LB1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail (ThermoFisher #78446) on a rotator at 4˚C for 10 minutes. Cells were then centrifuged, liquid decanted, and resuspended in Buffer LB2 (10mM Tris-HCL pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail. Cells were then centrifuged, liquid decanted and resuspended in ice cold Buffer LB3 (10mM Tris-HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-laurylsarcosine) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail. This nuclear resuspension was transferred into Covaris microtube 130 AFA Fiber pre-Slit snap-cap tubes (Covaris #520045) at a maximum concentration of 3 million cells per 130µL and sonicated using the following parameters on a Covaris ME220 according to the manufacturer's recommendations (Duration (s) = 360, Peak Power = 75, Duty Factor = 5.0, Cycles/Burst = 1000). Following sonication, 10µL of chromatin was removed for analysis of resulting DNA size and concentration. Briefly, 10µL of sonicated chromatin was incubated with 20µg RNase A (Thermo Scientific #EN0531) in TE pH 8 for 30 minutes at 37˚C in a thermal cycler. 20µg of Proteinase K (Thermo Scientific #EO0491) in Active Motif Elution Buffer AM4 (Active Motif #103926) was then added and the sample was incubated for 30 minutes at 55˚C followed by 90 minutes at 65˚C in a thermal cycler. Samples were then cooled to room temperature and DNA was purified using SPRIselect beads (Beckman #B23318) at a bead:sample ratio of 1.4:1 and eluted in 10µL of 10 mM Tris, 0.1 mM EDTA. Chromatin corresponding to 1 million cells was diluted with 200µL in Active Motif ChIP Buffer and incubated with pre-clearing Protein G agarose beads. After pre-clearing, agarose beads were pelleted and transferred to a new microcentrifuge tube containing 4µg of antibody specific to the target protein (AR: Abcam ab236225). IPs were then incubated overnight on a rotator at 4˚C. Protein G Agarose beads prepared the previous day according to Active Motif's Low Cell ChIP kit were added to the antibody-bound chromatin and incubated for 4 hours on a rotator at 4˚C. IPs were then transferred to ChIP filtration columns, washed, and eluted in 80µL according to Active Motif's Low Cell ChIP kit and DNA purified using SPRIselect beads at a bead:sample ratio of 1.4:1. After SPRI cleanup, ChIP samples and reverse crosslinked input DNA was made into sequencing Illumina libraries using Swift NGS 2S Plus (Swift Biosciences #21096) and indexed using Single Indexed Adapters with MIDs (Swift Biosciences #279384).  ChIP-seq libraries were prepared using Swift Biosciences Accel-NGS 2S Plus DNA Library Kit with UMIs.