GSM9005984: KP Tumor Control H3K9ac ChIP; Mus musculus; ChIP-Seq
Approximately 20x106 cells or  50-100 mg of pulverized tumors were crosslinked with 1% formaldehyde in PBS for 10-12 minutes at room temperature, quenched with 5 mg/mL bovine serum albumin in PBS and then washed twice with cold PBS. Cell pellets were resuspended in 1mL lysis buffer [50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail (Thermo Fisher Scientific)] and sonicated with a Covaris M220 sonicator for 25 minutes and 22 minutes for tumors and cell lines, respectively, to obtain chromatin fragment lengths of 100 to 1,000 bp. Fragments were evaluated by Bioanalyzer DNA High Sensitivity kit (Agilent). Fragmented chromatin was diluted in IP buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and incubated overnight at 4°C with Protein G magnetic beads (Dynabeads: Life Technologies) that had been pre-incubated with anti-H3K9ac (Abcam, ab4441) antibody. Immunoprecipitants were washed six times with wash buffer (50 mM HEPES pH 7.6, 0.5 M LiCl, 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40) and twice with TE buffer. Immunoprecipitated and IP input was treated with RNase A and Proteinase K on the beads, recovered in 1% SDS and 0.1 M NaHCO3 over a period of 6 h at 65 °C, and purified with DNA clean and concentrator-25 (Zymo Research). Up to 10 ng of DNA was used for the library construction using NEBNext Ultra II DNA Library Prep Kit (NEB, E7645). Sequencing was performed on NovaSeq (Illumina) for 150 nucleotides from paired ends according to the manufacturer's instructions.