GSM9218658: mES-E14-NANOG-FKBP-dTAG-6h_HA; Mus musculus; ChIP-Seq
ES cells were fixed for 30 min with 2 mM Di (N-succinimidyl) glutarate (DSG) (Sigma-Aldrich, 80424) followed by 30 minutes with 1% formaldehyde. Fixed cells were lysed in SDS lysis buffer (1% SDS (Sigma-Aldrich, 436143-25G), 10 mM EDTA (Sigma-Aldrich, 688363), 50 mM Tris-HCl, pH 8 with protease inhibitor cocktail (Abcam, b271306)) and the lysate was syringe-passaged through a 27-gauge needle. The cell lysates were sonicated using Covaris ME220 for 10 min. The sonicated lysates were pre-cleared with a 1:1 mix of Protein A and Protein G Dynabeads (Thermo Fisher 10001D / 10003D) for 15 minutes at 4°C. After three washes of the Dynabeads in PBS containing 0.01% tween-20 (Sigma-Aldrich, P6585-10ML), the sonicated samples were incubated overnight at 4°C. 5% of the lysate was set aside as input. After incubation, the Dynabead mix was added to each immunoprecipitate and rotated at 4 ̊C for 3 h. The beads were washed three times with RIPA buffer (50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once with TE-50 mM NaCl. The chromatin was eluted from the beads with SDS lysis buffer for 30 min at 65 °C, then treated with 1µL 500 ug/ml RNase A (Invitrogen, AM2286) for 30 min at 37 °C, followed by Proteinase K (Thermo Fisher, EO0491) 1µL 14-22 mg/ml overnight at 65 °C. DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo 5201). Library preparation for the Illumina platform was performed following the manufacturer's instructions (NEB, 7645S).