title: GSM1059387: H2A.Z; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-Seq of H3K122ac, H3K27ac, H2A.Z and H2A.Zac, chromatin was prepared by micrococcal nuclease digestion of crosslinked MCF-7 nuclei. For a typical experiment 3-6*10^7 MCF-7 cells were harvested, washed in PBS, resuspended in cold buffer I (300mM sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1mM EDTA pH 8.0, 15mM Tris pH 7.5, 1x protease inhibitors (Roche), 10mM sodium butyrate, 1mM DTT) at a density of 1.5x10^7 cells/ml. Cell suspension was mixed under gentle vortexing with the same volume of cold buffer II (as buffer I but with 0.5% NP-40) and incubated on ice for 10min to lyse the cell membrane. 8ml of a 40% sucrose cushion was prepared (buffer III: 1.2M sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1mM EDTA pH 8.0, 15mM Tris pH 7.5, 1x protease inhibitors, 10mM sodium butyrate, 1mM DTT) and a maximum of 3*10^7 cells were loaded onto one cushion. The released nuclei were separated from the cyctoplasm by centrifugation at 10,000rpm for 20min in a HB4 swing bucket rotor (Beckman) at 4°C.  MCF-7 cell nuclei were resuspendend in Mnase digestion buffer (320mM sucrose, 4mM MgCl2, 1mM CaCl2, 50mM Tris pH 7.5, 0.2mM AEBSF, 10mM sodium butyrate) with 0.2% formaldehyde and crosslinked at RT for 5min under constant agitation. The fixation was quenched by adding glycine to a final concentration of 25mM for 5min on a rolling wheel. Crosslinked nuclei were washed with Mnase digestion buffer once, pelleted again and resuspendend in Mnase digestion buffer with 60U/ml Mnase (Fermentas) and digested to >90% mononucleosomes at 37°C for 15min. Digestion was stopped by addition of 10mM EDTA final concentration and transferred to ice. After 15min on ice the samples were centrifuged at 10,000rpm for 10min and the supernatant containing the nucleosomes saved. 80µg of nucleosomes were diluted 1:10 in incubation buffer (50mM Tris pH 7.5, 75mM NaCl, 5mM EDTA pH 8.0, 0.1% NP40, 1x protease inhibitor, 10mM NaBut) and centrifuged at 13,000 rpm for 10min to remove any precipitant. Soluble nucleosomes were incubated with respective antibodies pre-bound to Dynabeads protein A (Invitrogen) at 4°C o/n. The immunocomplexes were collected in a magnetic rack and washed 1x in low salt buffer (50mM Tris pH 7.5, 150mM NaCl, 5mM EDTA pH 8.0, 1% Triton-X100, 0.5% NP-40) and 5x in high salt buffer (50mM Tris pH 7.5, 450mM NaCl, 5mM EDTA pH 8.0, 1% Triton-X100, 0.5% NP-40). After a final wash in TE buffer the immunocomplexes were eluted with 200µl N-ChIP elution buffer (incubation buffer with 1% SDS) in a thermoshaker at 30°C, 900rpm for 30min. The eluates were subsequently digested eith RNAse (Roche) and Proteinase K (Roche) and the DNA purified with the Qiaquick PCR purification Kit (Qiagen), according to the manufacturers instructions, and eluted in 80µl TE buffer. For Illumina sequencing the DNA from three individual immunoprecipitations was pooled. ChIP-Seq libraries were prepared according to manufacturer’s protocols and sequenced with Illumina GAII.