Mus musculus
Protocols: TDG -/- mouse embryonic stem cells were cultured on gelatinized plates in DMEM medium supplemented with 15% fetal bovine serum, 1% Penicillin Streptomycin, 1% MEM non-essential amino acids, 1% Glutamax, 50 μm β-mercaptoethanol and 103 U LIF, in normoxic conditions at 37°C. Tissues were collected from timed matings with the day of the copulation plug as E0.5. Embryos were dissected into hindbrain and heart. Nuclei (mESC) were treated with NaCNBH3 (80mM) in PBS for 3h at 37°C. Nuclei were sonicated for 8 min on Covaris M220 using the truChIP Chromatin Shearing Kit (Covaris) following the manufactures guidelined. Protein denaturation was achieved using binding buffer containing a chaotropic agent (from Genejet PCR purification kit, Thermofisher) followed by 4x 450 μL water washes in the Amicon Ultra-Spin column (30kDa cutoff) to obtain sheared DNA fragments. DNA libraries were obtained by end repair, A-tailing and adapter ligation (NEBNext Ultra II DNA library Prep kit) following the manufacturer's guideline. For ChIP, 100 μL dynabeads M-280 sheep anti-rabbit IgG were washed and preincubated with 10 μg antibodies (Upstate 06597, ab18255, ab1791 and ab8227) in PBS/BSA for 3 h at 4°C. Beads were subsequently washed with PBS/BSA and DNA libraries together with 1 ug salmon sperm DNA were added to the beads and incubated overnight at 4°C. Beads were then washed (5 min rotation at room temperature) 6x with 500 μL LiCl buffer (100 mM TRIS pH7.4, 500 mM LiCl, 1% NP40, 1% sodium deoxycholate) and 3x tube changes. For elution, beads were incubated (15 min rotation at room temperature) with 2x 100 μL elution buffer (0.1 NaHCO3, 1% SDS). To the combined 200 μL supernatant were then added 8 μL of 5 M NaCl, 4 μL of TRIS pH 7.4, 2 μL of 0.5 M EDTA and 1 μL proteinase K (10 mg/mL), incubated at 45°C for 1 h and purified using the Genejet PCR purification kit.
ENA first public
2018-10-22
ENA last update
2017-12-07
External Id
SAMEA104420725
INSDC center alias
Senior research associate
INSDC center name
Senior research associate
INSDC first public
2018-10-22T17:03:09Z
INSDC last update
2017-12-07T13:57:46Z
INSDC status
public
Submitter Id
E-MTAB-6271:h3_reduced_2
age
embryo
broker name
ArrayExpress
cell type
mouse embryonic stem cell
common name
house mouse
genotype
Tdg knockout
organism part
inner cell mass
sample name
E-MTAB-6271:h3_reduced_2
sex
not available