title: GSM7519232: T47D_Clone28_ER_dTAG_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: At the time of harvest, cells were fixed with 1% formaldehyde (Sigma) for 10 minutes at 37°C and quenched with 125mM Glycine (Fisher) for 10 minutes at 37°C. Plates were moved to ice, and cells were washed and scraped into ice cold PBS containing Complete EDTA-free Protease Inhibitor Cocktail (Roche). Cells were pelleted in aliquots of 3.6*107 cells, snap frozen in liquid nitrogen, and stored at -80°C. Pellets were thawed, and cells were lysed in 1mL Cell Lysis Buffer (85mM KCl, 0.5%NP40, 5mM PIPES pH 8.0), with protease inhibitor cocktail added fresh, for 10 minutes with rotation at 4°C. Nuclei were pelleted at 3300g at 4°C for 5 minutes and resuspended in 500μL ChIP lysis buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1), with protease inhibitor cocktail added fresh, for 10 minutes with rotation at 4°C. Lysates were moved to 15ml polystyrene conical tubes (Falcon) and sonicated in a Biorupter UCD-200 (Diagenode) on high for 30 seconds on and 30 seconds off for 4 sets of 5 cycles. Before each set, ice in the water bath was replaced, and samples were gently vortexed to mix. Sonicated lysates were then move to 1.5ml tubes and clarified by centrifugation at 14,000rpm for 15 min in 4°C. 500μL of the supernatant was diluted into 6.5mL Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 167mM NaCl, 16.6mM Tris-HCl pH 8.0), with protease inhibitor cocktail added fresh ( 1*106 cells in 200μL). 1ml ( 5*106 cells) was aliquoted into each of 3 tubes with antibody (1.25 μg anti-HA, Cell Signaling #3724S, 2.5μg anti-ER, Millipore #06-935, or 2.5μg IgG control, Cell Signaling #2729S), and incubated with end-over-end rotation at 4°C overnight. 50μL Protein A/G Magnetic Beads (Pierce) per sample were washed with bead washing buffer (PBS with 0.1% BSA and 2mM EDTA) and then incubated with samples for 2 hours with rotation at 4°C. The samples were washed once each with low salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 150mM NaCl, 20mM Tris HCl pH8.0), high salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 500mM NaCl, 20mM Tris Hcl pH 8.0), LiCl immune complex buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.0), and 1xTE (10mM Tris-HCl, 1mM EDTA pH8.0). Immune complexes were eluted in elution solution, (1% SDS, 0.1M sodium bicarbonate) in a thermomixer for 30 min at 65°C at 1,200rpm. Crosslinks were reversed and proteins were digested with the addition of 200mM NaCl and 2ul Proteinase K in a thermocycler at 65°C for 16 hours. DNA was purified with a Qiaquick PCR cleanup (Qiagen), and libraries were prepared with a NEBNext Ultra II Library Prep Kit (New England Biolabs).