title: GSM5230777: p53_IR_BaF3; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde in the culture media for 10 minutes at room temperature, followed by addition of 0.125M of glycine to stop fixation. Nuclei were extracted with 5 minutes lysis in 0.25% Triton buffer (10mM Tris-HCl pH8, 10mM EDTA, 0.5mM EGTA), followed by 30 minutes in 200mM NaCl buffer (10mM Tris-HCl pH8, 1mM EDTA, 0.5mM EGTA). Nuclei were resuspended in sonication buffer (10mM Tris pH8, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.5% SDS, 0.5% Triton X-100, 0.05% NaDOC) and sonicated for fourteen cycles of 30 seconds with a digital sonifier (Branson Ultrasonics) at 80%, with 30 seconds rest in cooled circulating water. Beads were prepared overnight with 40μL of Dynabeads Protein G (Invitrogen, Life Technologies) conjugated to 5μg of antibody against p53 (1C12, Cell Signaling Technology) or H3K27ac (ab4729, Abcam). Immunoprecipitation was performed by overnight incubation of antibody-bead matrices with sheared chromatin from the equivalent of 5x106 cells. Immune complexes were washed sequentially for 2 min at room temperature with 1 ml of the following buffers: wash B (1% Triton X-100, 0.1% SDS, 150-mM NaCl, 2-mM EDTA, and 20-mM Tris-HCl, pH 8), wash C (1% Triton X-100, 0.1% SDS, 500-mM NaCl, 2-mM EDTA, and 20-mM Tris-HCl, pH 8), wash D (1% NP-40, 250-mM LiCl, 1-mM EDTA, and 10-mM Tris-HCl, pH 8), and TEN buffer (50-mM NaCl, 10-mM Tris-HCl, pH 8, and 1-mM EDTA). The samples were de-crosslinked by overnight incubation at 65̊C in 1% SDS buffer (50mM Tris-HCl pH8, 10mM EDTA). Following RNaseA and Proteinase K enzymatic treatments, ChIP DNA was purified using Qiaquick PCR Cleanup kit (Qiagen). ChIP-Seq libraries were prepared using the Illumina TruSeq kit and sequenced on an Illumina HiSeq 2500 sequencer in paired-end 50bp configuration, with input DNA from the same cells sequenced as negative control (Input ChIP-Seq are submitted in GSE150667 and described in PMID: 32641579).