Illumina HiSeq 4000 paired end sequencing; ChIP-seq of H2A.Z and its histone chaperones during the cell cycle
ChIP-seq of H2A.Z and its histone chaperones during the cell cycle
Cells were detached with PBS supplemented with 125 mM EDTA.Cells were crosslinked in growth medium containing 1.2% v/v formaldehyde for 10 min, after which 125 mM glycine was added to stop the fixation process.  U2OS  cells were maintained in DMEM (high glucose) containing 10% v/v FBS, 1 mM sodium pyruvate, and 10 U/mL penicillin and streptomycin U2OS cells were stably transfected with 2 ug of pEXPR-IBA105-ANP32e, -H2A.Z, or -YL1 constructs using jetPRIME® transfection reagent (Polyplus-transfection® SA) according to the manufacturer's protocol. Antibiotic selection was performed using complete growth medium containing 3 mg/mL G 418. Cells were washed three times with ice-cold PBS and resuspended in 2 mL of ice-cold swelling buffer (25 mM HEPES pH 7.6, 15 mM NaCl, 10 mM KCl, 2 mM MgCl2, 0.2% v/v NP-40, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 M sodium butyrate) and incubated on ice for 10 min. This step was followed by addition of 2 mL of Solution II (0.6 M sucrose, 120 mM KCl, 15 mM HEPES pH 7.6, 15 mM NaCl, 2 mM MgCl2, 0.2% v/v NP-40, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 M sodium butyrate), and nuclei were released by Dounce homogenization using a B-type (tight) pestle. The cell suspension was overlaid onto 8 mL of Buffer III (sucrose cushion: 1.2 M sucrose, 60 mM KCl, 15 mM HEPES pH 7.6, 15 mM NaCl, 2 mM MgCl2, 0.2% v/v NP-40, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 uM sodium butyrate) and centrifuged at 3900 x g for 38 min to pellet nuclei. The nuclei pellet was resuspended in 670 uL of sonication buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 1% w/v SDS, 0.1% w/v sodium deoxycholate, 1% v/v Triton-X-100, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 uM sodium butyrate). The sample was sonicated in a Bioruptor on 'high' with 30 s on/off cycles for a total of 60 min and then centrifuged at 10,000 x g for 15 min. The supernatant containing small chromatin fragments was transferred to fresh tubes and the concentrations measured by absorbance at 260 nm using a NanoDrop spectrophotometer. For each pulldown reaction, 50 uL of uncoupled and Strep-Tactin Superflow high-capacity resin were used. A total of 1.3 mg of chromatin was used per reaction in pulldown buffer (50 mM Tris-HCl pH 7.6, 0.1 M NaCl, 1% v/v NP-40, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 M sodium butyrate). The sample was incubated with uncoupled beads for 1 h at 4 °C with rotation to remove nonspecific binders and centrifuged at maximum speed for 5 min, and the supernatant was transferred to Strep-Tactin beads and incubated overnight at 4 °C with rotation. The sample was washed three times for 5 min each with 900 uL of wash buffer (100 mM Tris-HCl pH 7.6, 0.15 M NaCl, 1 mM EDTA, 1x Roche EDTA-free protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitor cocktail, 5 uM sodium butyrate). Protein–DNA complexes and input chromatin were decrosslinked and eluted from the beads by incubation in 95 uL of Tris-EDTA (TE) (0.1:10) with 1 uL of RNase (Roche) for 30 min at 37 °C, followed by addition of 2.5 uL 20% w/v SDS and 2.5 uL Proteinase K (20 mg/mL), and incubation at 65 °C for 10 h. To purify decrosslinked chromatin, 100 uL of decrosslinked DNA was added to 170 uL of AMPure beads equilibrated to room temperature. The mixture was incubated at room temperature for 15 min to bind DNA and placed over a magnet for 2 min. The supernatant was removed and the beads were washed twice with 350 uL of 80% v/v ethanol. The beads were dried at room temperature. DNA was eluted with 50 uL of TE (0.1:10) and quantified using the Qubit™ dsDNA HS Assay Kit, as per the manufacturer's protocol.  Indexed libraries were prepared using a TruSeq DNA Nano kit (Illumina)