GSM8172218: 3T3_CDX2, CTCF, Dox, rep1; Mus musculus; ChIP-Seq
5 million cells were fixed in 1% formaldehyde for 10 min at room temperature, quenched with 250 mM Tris-HCl pH 8.0, washed with PBS, spun down, and stored at −80 °C. The cell pellet was resuspended in 1.5 ml LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% TritonX-100), incubated 10 min at 4 °C, spun down, and resuspended in 1.5 ml LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and incubated 10 min at 4 °C. The pellet was spun down and rinsed twice with SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS), and finally resuspended in 0.9 ml SDS shearing buffer. All buffers contain 1:100 diluted Protease Inhibitor Cocktail in DMSO (Sigma). The suspension was transferred to a milliTUBE 1 ml AFA fiber and sonicatedon a E220 focused ultrasonicator (Covaris) using the following settings: 20 min, 200 cycles, 5% duty, 140W, and input sample aliquots were taken.5μg of sonicated chromatin was incubated with 5μg CTCF antibody (#61932 Active Motif) and for normalization 50ng Drosophila chromatin (Active Motif) and 2.5μg anti-H2Av drosophila-specific antibody (Active Motif). ChIP was performed using ChIP-IT High Sensitivity kit (Active Motif) following manufacturer protocol. Libraries were prepared with NEBNext Ultra II DNA Library Prep Master Mix Set (NEB, #E7103) using insert size selection of 250 bp. Sequencing was performed using 37 nt paired end reads on an Illumina NextSeq 500.