GSM4040446: BMDM_LPS_H3K27Ac_rep2; Mus musculus; ChIP-Seq
For ChIP-Seq, 40 mio cells per sample 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/15 min 1% formaldehyde at room temperature. Remaining formaldehyde was quenched with 150 uM glycins, cells collected and washed 2x in ice-cold D-PBS. Pellets were stored at -80°C until ChIP was perormed. 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). Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).
GEO Accession
GSM4040446