title: GSM1890225: p53Mdm2ko_H2Ak119ub1 rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation (ChIP) was done according to the protocol published by Denissov and colleagues (Denissov et al., 2007). Briefly, cells were fixed in 1.1 % of PFA for 20 min, quenched with 0.125M glycine and lysed in 1% Triton X-100, 0,15 M NaCl, 1 mM EDTA, 0.3 % SDS and additional protease inhibitor. The cell lysate was sonicated as desired and used for antibody (2µg) and Protein A/G PLUS-Agarose incubation (Santa Cruz) over night. Bead interaction was released via 20 min rotation incubation in 1% SDS and 0.1 M NaHCO3 and DNA-protein crosslink was reversed via the addition of 0.2 M NaCl and shaking at 65 °C. DNA was purified by the MinElute PCR Purification Kit (Quiagen) and either used for targeted PCR (see primer table) or sequencing. For sequencing, DNA was sonicated again to reach fragment sizes less than 200 bp, and libraries were prepared with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB). The size range of final libraries was determined with the Bioanalyzer 2100 from Agilent (290-310 bp). Libraries were amplified and sequenced by using cBot.
identifiers.namespace: GEO
identifiers.id: GSM1890225