Illumina Genome Analyzer IIx sequencing; SPOC1 (PHF13) is a Molecular Reader and Effector of H3K4me2/3
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
ENA first public
2016-05-30
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA3143256
INSDC center alias
Max Planck Institute for molecular Genetics OWL Epigenomics
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
sex
male
Sequenced DNA Library
library_name
IgG Control
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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