GSM7065883: KO U2OS CENP-B ChIP replicate 3; Homo sapiens; ChIP-Seq
Cells were cross-linked at room temperature for 10 min using 1% formaldehyde. Cross-linking reaction was quenched by the addition of 125 mM glycine for 5 min, after which cells were washed in PBS. Cells were then resuspended in the lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 3mM CaCl2, 1 % Triton-X, 0.1% SDS, 0.1% Sodium Deoxycholate, Protease Inhibitors), and chromatin was digested by the micrococcal nuclease for 20-25 min at 37 °C. Reactions were stopped by the addition of 10mM EDTA and 20 mM EGTA, and then briefly sonicated. Samples were then cleared by centrifugation at 4 °C, and a fraction of the chromatin extract was saved for DNA purification (input). Chromatin extracts were diluted to 50 mM Tris pH 7.5, 250 mM NaCl, 1 % Triton-X, 0.1% SDS, 0.1% sodium deoxycholate, protease inhibitors), and incubated with CENP-B antibody for 1 h. Protein G Dynabeads (Life Technologies) were washed and added to the samples, which were then incubated overnight at 4 °C. Following immunoprecipitation, beads were pelleted using a magnetic rack, and washed 3 times in 50 mM Tris pH 7.5, 250 mM NaCl, 2 mM EDTA, 1 % Triton-X, 0.1% SDS, 5 times in 50 mM Tris pH 7.5, 500 mM NaCl, 2 mM EDTA, 1 % Triton-X, 0.1% SDS, once in 10 mM Tris-HCl pH 7.5, 0.25 M LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA, and once in 10 mM Tris pH 7.5, 1 mM EDTA. Beads were then resuspended in 10 mM Tris pH 7.5, 1 mM EDTA, 1 % SDS and incubated at 65 °C for 15 min. Eluted DNA and input samples were treated with RNase A at 37 °C for 30 min, and with Proteinase K at 65 C overnight in a buffer containing 50 mM Tris HCl pH7.5, 300 mM NaCl and 1% SDS. DNA was then isolated using SPRIselect beads (Beckman Coulter) and used for construction of libraries. Briefly, NEBNext Ultra DNA Library Prep Kit (New England Biolabs) was used for adaptor ligation. DNA was then purified using SPRIselect beads, and amplified for 15 cycles with Illumina indexing primers using NEBNext Ultra II kit and NEBNext Multiplex Oligos for Illumina kit as instructed by the manufacturer's protocol. Amplified libraries were cleaned using SPRIselect beads and used for sequencing on NovaSeq platform.