GSM9788056: anti-flag, astrocyte; Mus musculus; ChIP-Seq
Astrocytes were crosslinked first with 5 mM ethylene glycol bis (EGS) for 20 min at room temperature, followed by 1.11% formaldehyde for 10 min. Crosslinking was quenched in 140 mM glycine for 5 min at room temperature. Cells were washed in cold PBS and lysed in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl, 1% NP-40, 1% SDS, 0.5% sodium deoxycholate, supplemented with protease inhibitors. Chromatin was sheared to 200–500 bp fragments  and incubated overnight at 4 °C with anti-Flag or control IgG. Protein G agarose beads were added for 1 h at 4 °C, washed sequentially in low-salt buffer, high-salt buffer, LiCl buffer, and TE. Complexes were eluted in 1% SDS, 0.1 M NaHCO3. After reverse crosslinking, DNA was purified and ChIP–seq library preparation  was performed using NEBNext Ultra DNA Library Prep Kit.