GSM8328542: ChIP-seq_Input; Mus musculus; ChIP-Seq
The initial third of the small intestine (duodenum) was dissected and opened longitudinally to wash the inter-lumen space and expose epithelial cells. Tissue was cut into 1-inch pieces and rinsed with ice-cold PBS. Tissue was treated with 3mM EDTA/PBS for 25 minutes for mutant mice and 35 minutes for wild-type, with changes in EDTA solution at the initial 5- and 10-minute time points. Mechanical force was applied to release the epithelial cell layer from the underlying mesenchyme. Crypts and villi were separated with a 70uM filter. The smaller crypts pass through the pores of the filter while the larger villi stay on top. Villus cells were collected and washed twice with ice-cold PBS and pelleted by centrifugation at 300 rcf at 4°C. Cell pellets were cross-linked in 2 mM DSG (Thermo Scientific, 20593) for 45 min, and then further cross-linked in 1% formaldehyde (Sigma, F8775) for 20 min at room temperature, pelleted and washed with ice-cold PBS. Fixed cells were lysed in 3X volume of lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.0, 1X protease inhibitor cocktail (G-Biosciences, 786-433) and sonicated for 8 minutes (30sec on-off, 20% amplitude) using a QSonica Q800R3 sonicator to shear chromatin to 300-600bp. Protein A/G beads (Invitrogen, 10001D and 10004D) were washed with 1% BSA in PBS and pre-loaded with 5μg of anti-Esrra antibody (Santa Cruz sc-65718). Antibody-conjugated beads were incubated with sheared chromatin in dilution buffer with SDS at a final concentration of 0.24% on a rotator at 4°C overnight. Antibody-conjugated beads were collected by magnetic separation and washed five times with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Sodium deoxycholate, 1% NP-40, 0.5M LiCl), and once with TE buffer (10mM Tris, 0.1mM EDTA). Cross-links were reversed in 0.1M NaHCO3 and 1% SDS overnight at 65°C. DNA was column purified using the MinElute PCR purification kit (Qiagen, 28004) and quantified using Picogreen (Invitrogen, P7581). ThruPLEX DNA-Seq kit (Takara, R400675)