GSM3406738: WT MEF 2 days H3K4me3 A1; Mus musculus; ChIP-Seq
Cells were fixed with PBS 1% formaldehyde. Fixation was quenched with 0,125M glycine. Cells were collected in SDS Buffer (100mM NaCl, 50mM Tris-HCl pH 8.1, 5mM EDTA pH 8, 0.5% SDS), centrifuged, then resuspended in 3 ml of ice-cold IP buffer (2 volumes SDS Buffer: 1 volume Triton Dilution Buffer) (Triton Dilution Buffer: 100mM NaCl, 100mM Tris-HCl pH 8.6, 5mM EDTA pH 8, 5% Triton X-100) and sonicated through the Digital Sonifier 450 (Branson) (ChIP-seq for Menin: 700-bp DNA fragments; ChIP-seq for H3K4me3 and H3K27me3: 200-bp DNA fragments). The immunoprecipitated product was purified through Protein G dynabeads (ThermoFisher Scientific, 10003D), afterwards washed with low-salt (150mM NaCl, 20mM Tris-HCl pH 8, 2mM EDTA pH 8, SDS 0.1%, 1% Triton X-100) and high-salt wash (500mM NaCl, 20mM Tris-HCl pH 8, 2mM EDTA pH 8, SDS 0.1%, 1% Triton X-100) buffer 3 times with 1 ml of 150 mM Wash Buffer and once with 1 ml of 500 mM Wash Buffer with the use of a Dynamag magnet (ThermoFisher Scientific, catalog number 12321D). Decrossilinking occurred through the incubation of beads (and the 1% input) with Decrosslinking Buffer (1% SDS, 0.1M NaHCO3) at 65 °C overnight. DNA was purified with the QiaQuick PCR Purification kit (Qiagen) following manufacturer's instructions. Libraries were prepared as previously described in Adamo et al., Nature Genetics 2015.