GSM6938271: input_CM; Mus musculus; ChIP-Seq
Cells under the reported conditions were fixed with 1% Formaldehyde (Cat. No. 252549, Sigma) for 5' RT. Then cells were washed three times with ice cold PBS 1X. Cells were lysed with 900 ul of buffer 1 (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 0.1% Nonidet P-40, 10% glycerol and protease and phosphatase inhibitors) for 8 min' at 4°C. This buffer is needed to extract cytoplasmic proteins. The samples were centrifuged at 3000 rpm for 5' at 4°C. Then the pellet containing nuclei is lysed with 500 ul of buffer 2 (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 1% SDS and protease and phosphatase inhibitors). Nuclear extracts were sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 0.5-2 kbp. Lysates were incubated overnight at 4°C shaking with Acetyl-Histone H3K9 antibody (#9649, Cell signaling, 1:50). The day after the immunocomplex were incubated at 4°C rotating with protein A beads (Ge 17-0780-01) for 1.5 h. After incubation, beads were washed thoroughly and then centrifuged. The chromatin was eluted from the beads, and cross-links were removed by incubation at 56°C for 12 hours. DNA was then purified using the DNeasy Blood & Tissue Kit (Cat. No. 69504, Qiagen) according to the manufacturer's instructions ChIP and input DNA (50 ng) were used for indexed library preparation using NEBNext Ultra II DNA Library Prep kit and subjected to 75 bp single-end sequencing on the Illumina NextSeq500.