ChIP Seq of Oct4, Nanog, H3K27me3 in Oct4+/+ and Oct4 +/- mES cells
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
2013-05-07
ENA last update
2018-03-08
External Id
SAMEA1886768
INSDC center alias
GIS
INSDC center name
Genome Institute of Singapore
INSDC first public
2013-05-07T17:03:04Z
INSDC last update
2018-03-08T16:22:46Z
INSDC status
public
Submitter Id
E-MTAB-1617:Input_Oct4+-mESC
broker name
ArrayExpress
cell line
OKO160
cell type
embryonic stem cell
common name
house mouse
genotype
Oct4+/-
sample name
E-MTAB-1617:Input_Oct4+-mESC
Sequenced DNA Library
library_name
Input_Oct4+-mESC
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ESCs were cultured as described in GMEM/10% FCS with 100 units/ml LIF on gelatinised tissue culture flasks at a density of 2.5-10x104/cm2. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ESCs were fixed with 1% formaldehyde (10 min, rt) and crosslinking stopped by addition of glycine to 0.2 M. Cells were lysed by incubating in 10 mM Tris-HCl, 0.25% Triton X-100, 10 mM EDTA, 100 mM NaCl (twice, 15min, 4 C). Nuclei were lysed in 50 mM HEPES-KOH, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 1% SDS (15 min, 4 C), centrifuged and the chromatin pellet resuspended in 0.1% SDS buffer for shearing on ice to an average size of about 250 bp using a probe sonicator (Branson digital sonifier S-450D). Chromatin extracts were pre-cleared with Protein G Dynal Magnetic Beads (Invitrogen) (2 h, 4 C) and immunoprecipitated overnight at 4 C using Protein G Dynal Magnetic Beads pre-coupled with antibodies against Oct4 (Santa Cruz N19, sc8628), Nanog (Cosmo Bio RCAB0002P-F) or H3K27me3 (Millipore 07-449). Beads were washed 3x with 0.1% SDS buffer, once with 0.1% SDS/ 0.35M NaCl buffer, once in 10 mM Tris-HCl, 0.25M LiCl, 1 mM EDTA, 0.5% deoxycholate, 0.5% NP-40, and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Immunoprecipitated material was eluted from the beads and crosslinks reversed by incubation with pronase for 2 h at 42 C then 6 h at 67 C. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ChIP-Seq library was prepared using the ChIP-Seq Sample Preparation Kit (Illumina) and NEBNext?? ChIP-Seq Library kit (NEB Biolabs) according to the manufacturer's instructions and sequenced for 36 cycles with the HiSeq 2000 system (Illumina).