GSM5343301: ZFP423H1285N-Avi_input_1; Mus musculus; ChIP-Seq
iWAT SVF isolated from Control (Cre negative), Adipo-Zfp423WT-Avi or Adipo-Zfp423H1285N-Avi mice were induced to differentiate in vitro. Differentiated adipocytes were treated with 5 uM DOX for 12 hr to induce BirA expression and biotinylation of ZFP423 proteins. 48 hr after DOX treatment, cells 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. Fixed 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, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, and Protease inhibitor cocktail [Sigma, #P8340]) and pre-cleared using Streptavidin Sepharose High Performance beads (GE Healthcare Bio-sciences, #17-5113-01) for 1 hour at 4 °C. Pre-cleared samples were incubated with the streptavidin sepharose beads at 4 °C for 2 hours. Affinity purified material was consecutively washed with SDS wash buffer (2% SDS in 50 mM Tris-HCl pH 7.9 and Protease inhibitor cocktail [Sigma, #P8340]) twice, high salt wash buffer (50 mM Tris-HCl pH 7.9, 1mM EDTA, 500 mM NaCl, 0.1% sodium deoxycholate, 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, 0.5% NP-40, 0.5% sodium deoxycholate, and Protease inhibitor cocktail [Sigma, #P8340]), and 1x Tris-EDTA (TE). After elution at SDS elution buffer (50 mM Tris-Hcl pH 7.9, 1% SDS, and 10mM EDTA) at 70°C overnight, the affinity purified material was digested with RNase (Roche, #11119915001) and proteinase K (ThermoFisher Scientific, #EO0491) prior to the purification and concentration of the affinity purified genomic DNA by ChIP DNA Clean & Concentrator kit (Zymo Research, #D5201). ChAP-isolated DNA was subjected to library production (ChAP-seq) using NEBnext NGS DNA Library Preparation for Illumina kit (New England BioLabs, #E7645). Sequencing was performed with Illumina NextSeq 500 Mid Output (130M) by the UT Southwestern McDermott Center Next Generation Sequencing Core. DNA libraries were prepared for sequencing using NEBNext NGS DNA Library Preparation for Illumina Kit following manufacturer's instructions.
GEO
GSM5343301