Mus musculus
Protocols: E14 ES cells were grown in the presence of LIF and 20% FBS. The cells were collected and cross-linked for 15 minutes with 1% formaldehyde. The fixation reaction was quenched by the addition of glycine (0.125M) for 10 minutes. The cells were then lysed on ice for 10 minutes in 1.3 ml of ChIP buffer, 1 volume of SDS buffer (100mM NaCl, 50 mM Tris-HCl pH8, 5mM EDTA, 0.2% NaN3 and 0.5% SDS) : 0.5 volume of Triton dilution buffer (100mM Tris-HCl pH8.6, 100 mM NaCl, 5 mM EDTA, 0.2% NaN3 and 5% Triton-X 100). The lysate was passed through a syringe multiple times and then sonicated in Bioruptor at high frequency for 2 x 6 cycles. The lysate was then centrifuged at 13,000 rpm for 30 minutes to pellet debris and a protein determination was measured. 1 mg of chromatin was then incubated with 5 µl of SPOC1 antibody (CR56) or rabbit IgG control antibody overnight at 4°C under rotation. 30 µl of protein A beads were then added to the reaction and allowed to incubate at 4°C for 2h. The beads were then washed by successive low salt (0.1% SDS, 1% Triton X, 2 mM EDTA, 20 mM Tris-HCl and 150 mM NaCl), high salt (0.1% SDS, 1% Triton X, 2 mM EDTA, 20 mM Tris-HCl and 500 mM NaCl) and LiCl washes and then eluted for 3h at 65°C with 110 µl of elution buffer (1% SDS and 100 mM NaHCO3). The RNA and proteins were then digested with RNAse A and Proteinase K and the DNA purified and sequenced Library construction;according to manufacturers protocols
ENA-FIRST-PUBLIC
2016-05-30T17:04:33Z
ENA-LAST-UPDATE
2018-03-08T22:13:02Z
External Id
SAMEA3143256
INSDC center name
Max Planck Institute for molecular Genetics OWL Epigenomics
INSDC first public
2016-05-30T17:04:33Z
INSDC last update
2018-03-08T22:13:02Z
INSDC status
public
Submitter Id
E-MTAB-2636:IgG Control
broker name
ArrayExpress
cell line
ES-E14 cell
cell type
embryonic stem cell
common name
house mouse
sample name
E-MTAB-2636:IgG Control
scientific_name
Mus musculus
sex
male