Approximately 10x107 Cells were used per ChIP. Cells were trypsinized, resuspended in 40 ml of ES cell medium containing 1% Methanol-free Formaldehyde (Thermo/Pierce) and incubated for 10 min on a rotor at room temperature (RT). Then, Glycine solution was added to a final concentration of 125 mM and incubated for 5 min on a rotor at RT. Next, the cells were centrifuged at 2000 rpm for 10 min at 4°C and washed two times with 50 ml of cold PBS. The cell pellet was resuspended in 10 ml of lysis buffer 1 (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) and then incubated on a rotor for 10 min at 4 °C. The cells were then pelleted, resuspended in 10 ml of lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors) and incubated at RT for 10 min on a rotor. The cells were pelleted again and resuspended in 2ml of sonication buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100, 1× protease inhibitors). The suspension was trasnfered to 1ml AFA tubes (Covaris) and sonicated with the following settings: 140 peak incidence, 5 duty factor, 200 cycles/burst, 1200 secs in a E220 Ultrasonicator (Covaris). The sonicated lysate was recovered into DNA LoBind tubes (Eppendorf) and centrifuged at 16,000 g for 10 mins at 4°C to eliminate debris. Meanwhile, 100ul of GammaBind G Sepharose (Sigma) beads were washed twice and equilibrated for 15h at 4°C in blocking solution (0.5% BSA in PBS). The sonicated lysate was pre-cleared for 3h at 4°C with 100 ul of blocked beads equilibrated in sonication buffer. The samples were centrifuged for 3 mins at 16,000 g to recover the precleared lysate, 10% of the total volume was set aside as input control. Each ChIP was set up by adding 10ug of antibody (Supplementary table) to the lysate and incubating it 15h at 4°C on a rotator. The beads were then washed once with the sonication buffer, once with washing buffer 1 (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100), once with washing buffer 2 (10mM Tris-HCl pH8, 250mM LiCl, 2mM EDTA, 1% NP40), once with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 50mM NaCl, and twice with TE buffer. Following washing, the beads were resuspended in 100 µl of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1.0% SDS) and incubated at 65°C for 30min at 800rpm. The supernatant was transferred to a new tube and further 100ul of elution buffer were added to the beads to repeat the elution and recover any remaining complexes. The input and eluates were then incubated first with 0.1 mg/ml RNase A (Thermo) at 37°C for 2h, and then with 0.1 mg/ml of Proteinase K (NEB) at 55°C for 2h. Finally, the DNA was purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer instructions, the samples were eluted in 50 µl of milliQ water and stored at -20 °C until analysis. To generate libraries, DNA was first subjected to 300-1000 bp size selection with Agencourt AMPure XP beads (Beckman Coulter). Libraries were generated using the ChIP-Seq Ultra DNA Library Prep Kit for Illumina (NEB). Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina HiSeq2500. Library preparation and sequencing were performed by the Deep Sequencing Facility of the Max-Planck Institute of Immunobiology and Epigenetics.
GEO Accession
GSM4456466