mESC_Zscan10-FKBP12(F36V)_cloneC2_dTAGv1
Protocols: 30 million mESCs were detached with 50,000 mESCs were detached with Accutase (Innovative Cell Technologies) and were cross-linked with 1% (vol/vol) formaldehyde in PBS for 10 min, and the reaction was quenched with Tris-HCl (200 mM, pH = 8). Mouse embryonic stem cells (mESCs) were cultured in 2i+LIF medium composed of DMEM (Thermo Fisher, 10566-024), 10% ES Cell FBS (Thermo Fisher, 16141079), 1X MEM Non-Essential Amino Acids Solution (Thermo Fisher, 11140050), 1 mM sodium pyruvate (Thermo Fisher, 11360-039), 100 µM 2-Mercaptoethanol (Thermo Fisher, 31350-010), 100 U/ml Penicillin-Streptomycin (Thermo Fisher, 15140-122), 100 ng/ml of mouse LIF (Protein Production and Structure Core Facility, EPFL), 3 μM of GSK3 inhibitor (CHIR99021; Tocris Biosciences, 4423), and 2 μM of MEK1/2 inhibitor (PD0325901; Tocris Biosciences, 4192). To induce the degradation of Zscan10 endogenously tagged with an FKBP12(F36V), the cells were cultured in medium containing either 500 nM dTAGv-1 (MedChemExpress, HY-145514) or the same volume of DMSO for 24 h prior to harvest. The cross-linked nuclei were extracted and sonicated with the Covaris E220 Focused-ultrasonicator with the following settings: 20 min at 5% duty cycle, 140 W, 200 cycles.  The fragmented chromatin was on-bead tagmented with pA-Tn5 (Protein Production and Structure Core Facility, EPFL) on a thermomixer at 37°C for 5 min with 800 rpm shaking. The chromatin was washed twice with low-salt wash buffer and was de-crosslinked with 1 µl Proteinase K (20 mg/ml) at 65°C and 1,400 rpm shaking overnight. DNA was purified with MinElute PCR purification kit (Qiagen, 28006) and was PCR amplified with Q5 High-Fidelity 2X Master Mix (New England Biolabs, M0492L) and indexed primers. The amplified library was cleaned-up by adding 1.2 times the volume of AMPureXP beads (Beckman Coulter, A63882) and eluted with 10 mM Tris-HCl, pH 8.0.
isolate
not applicable
organism
Mus musculus
organism
Mus musculus
cell_line
ES cell line
collection date
not collected
scientific_name
Mus musculus
common name
house mouse
genotype
Zscan10-FKBP12(F36V)
geographic location (country and/or sea)
not collected