GSM8529210: antiNrf1_control_1; Homo sapiens; ChIP-Seq
After treatment, cells were fixed with 1% formaldehyde 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 2*10^7 cells, snap frozen in liquid nitrogen, and stored at -80°C. Pellets were thawed, and cells were lysed in 1mL ChIP Lysis Buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCL pH 8.0), with protease inhibitor cocktail added fresh, for 10 minutes with rotation at 4°C. Lysates were sonicated at 70% amplitude for 15 seconds on and 45 seconds off for 4 sets of 20-minute cycles. Sonicated lysates were moved to 1.5ml tubes and clarified by centrifugation at 14,000rpm for 10 min in 4°C. 50μL of the supernatant was diluted into 760μL ChIP 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*10^6 cells in 200μL). 1ml (4*106 cells) was aliquoted into each of 3 tubes with antibody (7.32μg anti-NRF1 Invitrogen MA5-35366 or 3μg anti-YY1 Invitrogen 703054), and incubated with end-over-end rotation at 4°C overnight. 80μL Protein A/G Magnetic Beads (New England Biolabs) per sample were washed with bead washing buffer (PBS with 0.1% BSA and 2mM EDTA) and then incubated with samples for 90 minutes 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). 1μl RNase A was added to each sample for 10 min at 37°C. Proteins were digested with the addition of 5μl Proteinase K and incubated in a 65°C water bath overnight. DNA was purified with a MinElute PCR purification kit (Qiagen), and libraries were prepared with a NEBNext Ultra II Library Prep Kit (New England Biolabs).