GSM8215289: 182_27ac_S1; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde (catalog#F8775, sigma) for 10 minutes at room temperature. The reaction was stopped by adding glycine to a final concentration of 125 mM. Cells were washed with PBS and lysed in cell lysis buffer (50mM Tris-HCl, pH 8.0, 100mM NaCl, 5mM EDTA, 0.5% SDS) containing 1X Protease Inhibitor Cocktail (catalog#A32955, ThermoFisher) for 10 minutes.  This was spun at 800x G at 4°C for 5 minutes and the cell pellet was resuspended in 0.5 ml of Nuclear Lysis Buffer (50mM Tris-HCl, pH 8.0, 10mM EDTA, 0.2% SDS). Lysates were sonicated to yield 200–500 bp DNA fragments and diluted with ChIP dilution buffer (16.7mM Tris-HCl, pH 8.0, 167mM NaCl, 1.2mM EDTA, 1.1% Triton-X100). 1:100 of anti-H3K27ac (catalog#8173; CST), anti-HMGN1 antibody (catalog#12734S; CST), or anti-menin antibody (catalog#A300-105A; Fisher Scientific) were added to the sample and gently mixed overnight at 4°C. 50 μL of Pierce™ ChIP-grade Protein A/G Magnetic Beads (catalog#26162; Thermo Fisher) were washed with ChIP dilution buffer and gently mixed with the sample for 2 hours. Beads were washed with low-salt wash buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl, 2mM EDTA, 1% Triton-X100, 0.05% SDS), high-salt wash buffer (20mM Tris-HCl, pH 8.0, 500mM NaCl, 2mM EDTA, 1% Triton-X100, 0.05% SDS), LiCl wash buffer (10mM Tris-HCl, pH 8.0, 250mM LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA), and TE buffer pH 8.0 (catalog# AM9858; Thermo Fisher). Immunoprecipitated DNA was eluted with 100uL elution buffer (1% SDS, 100mM NaHCO3, 10mM DTT), added with 1ul proteinase K, incubated at 62°C for 2-8 hours with shaking. This was followed by an incubation at 95°C for 10 minutes followed by cooling and PCR purification. DNA was purified using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit (catalog#NC0389462). Library preparation and sequence were performed by Integrated Genomics Operation (IGO) core facility at MSKCC. Library was prepared using KAPA HTP library kit (KAPA biosystems).