GSM6280761: BJ1, Input; Mus musculus; ChIP-Seq
BJ1 and JB1 ESCs for ChIP-seq experiment were fixed for 5 min in 2% formaldehyde at room temperature while tumbling. A 15 million cell pellet was sequentially lysed in the following 3 buffers: 5mL Buffer 1 (10% Glycerol, 50 mM Tris pH7.5, 140 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.25% Triton X100, 0.33X protease inhibitors (Roche)) for 10 min on ice; 5mL Buffer 2 (10 mM Tris pH7.5, 0.2 M NaCl, 1 mM EDTA, 0.33X Protease inhibitors (Roche)) for 10 min on ice; 1mL Buffer 3 (100 mM NaCl, 10 mM Tris pH7.5, 1 mM EDTA, 0.25% SDS, 0.1% Sodium Deoxycholate, 1X Protease inhibitor (Roche)) for 10 min on ice. The chromatin was then sheared for 15 min using a Covaris S220 device (Peak power: 140W; Duty Factor: 20%; Cycle/Burst: 200), clarified by centrifugation, quantified and stored at -80°C.  For CTCF ChIP-seq experiment, 10 µg sonicated chromatin was diluted in the following buffer (16 mM Tris pH8, 1 mM EDTA, 167 mM NaCl, 1.1% Triton X100, 0.01% SDS, 1X Protease inhibitor (Roche)) to a total volume of 1mL and was precipitated with 5 µg CTCF antibody (Millipore # 07-729) overnight. After recovering the antibody-bound chromatin with Salmon Sperm DNA/Protein A agarose beads (Millipore #16-157), beads were sequentially washed in Low Salt Wash buffer (20 mM Tris pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X100, 0.1% SDS), High Salt Wash buffer (20 mM Tris pH8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X100, 0.1% SDS), LiCl Wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.26 M LiCl, 1% NP-40, 1% Na Deoxycholate) and twice in TE buffer (10 mM Tris pH7.5, 1 mM EDTA). Chromatin was then eluted in 1% SDS, 0.1 mM NaHCO3 and decrosslinked over-night. ChIP-seq libraries were prepared on purified DNA using the NEB Ultra II DNA library prep kit following manufacturer's instructions