Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For ChIP-Seq, 40 mio cells per sample or 20 mio cells in case of H3K4me1/me2/me3 were fixed as mentioned in the sample section either for 30 min in 2 mM DSG (#C1104 ProteoChem) and 10 min 1% MeOH-free formaldehye (#2890, ThermoFisher) or 10 min 1% formaldehyde at room temperature. Remaining formaldehyde was quenched with 150 uM glycin, cells collected and washed 2x in ice-cold D-PBS. Pellets were stored at -80°C until ChIP was performed. For ChIP-Seq nuclei were extracted in Fast-IP buffer (150 mM NaCl, 5 mM EDTA (pH=7.5), 5 mM Tris (pH=7.5), 1% Triton X-100, 0.5% NP40), chromatin sonicated in shearing buffer (1% SDS, 10 uM EDTA (pH=8),50 mM Tris (pH=8)) using the Bioruptor Pico (Diagenode) for 10-15 cycles (30s on/off) at high settings. Sonicated chromatin was diluted 1:10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 uM EDTA (pH= 8), 16.7 uM Tris (pH=8), 167 mM NaCl) and the immunoprecipitation against the protein of interest was performed over night at 4°C. Antigen-antibody complexes were captured with protein A/G Dynabeads (Life Techn. #11202D or #11204D), 5x washed in Fast-IP buffer. Reverse crosslink was performed overnight at 65°C and proteins degraded with 100 uM proteinase K. The DNA was puriefied using Quiagen PCR purification kit according to manufacture?s manual (#28006).