National Research Center for Epigenome Reprogramming Network
INSDC first public
2015-06-30T17:01:04Z
INSDC last update
2018-03-08T18:24:58Z
INSDC status
public
Submitter Id
E-MTAB-2002_AddSamples:cell_culture_1
broker name
ArrayExpress
cell type
mouse embryonic stem cell
common name
house mouse
sample name
E-MTAB-2002_AddSamples:cell_culture_1
scientific_name
Mus musculus
specimen with known storage state
fresh specimen
Sequenced DNA Library
library_name
H3K27me3
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
We purchased normal mouse embryonic stem cells (E14) from the Mutant Mouse Regional Resource Centers (MMRRC). We cultured stem cells in DMEM containing 15% (v/v) FBS (Hyclone), 2 mM L-glutamine, 1% (v/v) nonessential amino acids, 55uM-mercaptoethanol, antibiotics (all from Gibco) and 1,000 U ml-1of ESGRO (Chemicon) on gelatin-coated plates. ESC differentiation was induced by LIF withdrawal of ESC medium in monolayer cultures. PLKO-puro constructs expressing shRNAs were purchased from Sigma. The shRNA constructs targeting Ctbp2 are TRCN0000109335 and TRCN0000307495. Lentiviruses were produced with 293FT cell by co-transfection of 1 mg each of pMD2.G, pMDLg/pRRE, pRSV-rev, pLKO-shRNA using Lipofectamin (Invitrogen). 48 h after transfection, virus-containing medium was collected and filtered by 0.45 mm filters. Polybrene (10 mg ml-1) was added just before infection to target cells and infection was performed for 5 h. Puromysin selection (2 mg ml-1) was performed 48 h after infection for 2 days. As a control, we used pLKO.1 puro vector. Cells were cross-linked with 1% (v/v) formaldehyde for 10min at 37C and lysed with buffer containing 85mM KCl, 5mM PIPES (pH 8.0) and 0.5% (v/v) NP-40. After centrifugation, nuclear pellets were resuspended in buffer containing 10mM EDTA, 100mM Tris-Cl (pH 8.1) and 1% SDS. To solubilize and shear cross-linked DNA, lysates were sonicated. 10-fold diluted lysates in IP buffer containing 16.7mM Tris-Cl (pH 8.1), 167mM NaCl, 0.01% (w/v) SDS, 1.1% (v/v) Triton X-100 and 1.2mM EDTA were incubated overnight at 4C with protein A/G PLUS agarose and 1-2ug appropriate antibodies.(Ctbp2 antibody=BD Transduction Laboratories, cat#612044; normal mouse igG antibody = Santa Cruz, cat#sc-2025) Beads were washed with 1X 20mM Tris-Cl (pH 8.1), 150mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100, 2mM EDTA, 1X 20mM Tris-Cl (pH 8.1), 150mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100, 2mM EDTA, 1X 0.25M LiCl, 1% (v/v) NP40, 1% (v/v) Deoxycholate, 1mM EDTA, 10mM Tris-Cl (pH 8.1) and 2X with TE. Immune complexes were then eluted by adding 250ul of elution buffer containing 1% (w/v) SDS and 0.1M NaHCO3 two times. Cross-linking was reversed by adding of 20ul of 5M NaCl and incubated overnight at 65C. DNA was precipitated with ethanol and kept in -20C for the further use.