mESC_Zscan10-FKBP12(F36V)_cloneC2_dTAGv1
Protocols: 50,000 mESCs were detached with Accutase (Innovative Cell Technologies). 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. Nuclear extraction and permeabilisation were performed on the detached cells, followed by 2.5 µl Tn5 (Illumina, 20034197) tagmentation in a 50 µl reaction at 37°C for 30 min on a thermomixer with 1,000 rpm mixing. The transposed DNA was cleaned-up with DNA Clean and Concentrator-5 Kit (Zymo Research, D4014). The cleaned DNA was PCR amplified with Q5 High-Fidelity 2X Master Mix (New England Biolabs, M0492L) and indexed primers ordered from IDT. The amplified library was double size-selected by 0.5 and 1.5 volumes of AMPureXP beads (Beckman Coulter, A63882) to enrich for fragments ranging from 150-700 bp and was 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