GSM5343326: KO_CHD4_IP_2; Mus musculus; ChIP-Seq
Treated differentiated adipocytes were cross-linked with 1% formaldehyde in PBS for 10 minutes at 37 °C and quenched in 125 mM glycine in PBS for 5 minutes at 4 °C. DPP4+ cells were then lysed in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]) to obtain nuclear material. Crude nuclear pellets were collected by centrifugation and then lysed by incubation in lysis buffer containing 5 mM Tris-HCl pH 7.9, 1% SDS, 10 mM EDTA, 1mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]. Chromatin fragmentation (200-500 bp length) was performed at 4 °C by Bioruptor 300 using the setting of 10 cycles of 30 seconds on and 60 seconds off. Soluble chromatin was diluted 1:10 with dilution buffer (20 mM Tris-HCl pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM DTT, and Protease inhibitor cocktail [Sigma, #P8340]) and pre-cleared using Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-sciences, #17-0618-01) for 1 hour at 4 °C. Pre-cleared samples were incubated with the indicated antibodies at 4 °C overnight. Antibody-protein-DNA complexes were captured by incubation with Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-sciences, #17-0618-01) at 4 °C for 2 hours. Immunoprecipitated material was consecutively washed with low salt wash buffer (20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, and Protease inhibitor cocktail [Sigma, #P8340]), high salt wash buffer (20 mM Tris-HCl pH 7.9, 2mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, and Protease inhibitor cocktail [Sigma, #P8340]), LiCl wash buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, and Protease inhibitor cocktail [Sigma, #P8340]), and 1x Tris-EDTA (TE). After elution (100 mM NaHCO3, 1% SDS), the immunoprecipitated material was digested with RNase (Roche, #11119915001) and proteinase K (ThermoFisher Scientific, #EO0491) prior to the purification and concentration of the immunoprecipitated genomic DNA by ChIP DNA Clean & Concentrator kit (Zymo Research, #D5201). 50ng immunoprecipitated DNA of each sample was used for the construction of sequencing libraries. DNA libraries were prepared for sequencing using NEBNext NGS DNA Library Preparation for Illumina Kit following manufacturer's instructions.
GEO
GSM5343326