Illumina HiSeq 2500 sequencing; ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in EpiSCs undergoing reprogramming
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
EpiSC
NA
NA
Attributes by original data submitter
Sample
ENA first public
2018-06-01
ENA last update
2016-12-13
ENA-CHECKLIST
ERC000011
External Id
SAMEA26419918
INSDC center alias
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine
INSDC center name
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine
INSDC first public
2018-06-01T17:03:51Z
INSDC last update
2016-12-13T10:42:03Z
INSDC status
public
Submitter Id
E-MTAB-5342:Esrrb, 8 h
broker name
ArrayExpress
cell type
epiblast stem cell
common name
house mouse
genotype
Esrrb-tet-on
growth condition
K10+2i/LIF
sample name
E-MTAB-5342:Esrrb, 8 h
Sequenced DNA Library
library_name
Esrrb, 8 h_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Tet-on Esrrb EpiSCs (GOF18-E3 EpiSCs carrying the Tet-on-Esrrb-IRES-Venus and Pou5f1 [3.5 kb upstream of the TSS]-IRES-Puro transgenes) were maintained in mouse embryonic fibroblast-conditioned media (MEF-CM) containing 20% KnockOut Serum Replacement (KSR) supplemented with 10 ng/ml FGF2 (CM+FGF2) on fetal bovine serum (FBS)-coated dishes. For reprogramming, Tet-on Esrrb EpiSCs were dissociated into single cells using Accutase and plated in CM+FGF2 supplemented with 5 μM Y-27632 on FBS-coated dishes. After overnight culture, the medium was replaced by DMEM/F12 containing 10% KSR supplemented with LIF, 2i (1 μM PD0325901 and 3 μM CHIR99021), and 1 μg/ml Dox. Cells were harvested and cross-linked with 1% formaldehyde in PBS for 10 min at room temperature, and chromatin was sheared using Diagenode Bioruptor (high power, 40 cycles of 30 sec on and 30 sec off) equipped with a water-cooling system (4°C) in sonication buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 0.5% SDS) supplemented with 1× protease inhibitor cocktail (Sigma). Sheared chromatin with an average fragment size of 200‒300 bp was immunoprecipitated in 4× volume of ChIP dilution buffer (10 mM Tris-HCl [pH 8.0], 125 mM NaCl, 0.125% sodium deoxycholate, 1.25% Triton X-100) using Dynabeads protein A or G coupled with the following primary antibodies: mouse anti-Esrrb (PP-H6705-00, R&D Systems); rabbit anti-Nanog (A300-397A, Bethyl Laboratories); goat anti-Oct4 (sc-8628, Santa Cruz); goat anti-Sox2 (GT15098, Neuromics). Beads were washed once with low-salt buffer (20 mM, Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with high-salt buffer (20 mM, Tris-HCl [pH 8.0], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with RIPA buffer (50 mM, Hepes-KOH [pH 7.6], 250 mM LiCl, 1 mM EDTA, 1% Igepal CA630, 0.7% sodium deoxycholate), and once with TE containing 50 mM NaCl. After incubation with elution buffer (10 mM, Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% SDS), immunoprecipitated and input DNA were reverse cross-linked and purified. Sequencing libraries were prepared according to the TruSeq ChIP Sample Preparation protocol (Illumina) with size selection of 250‒450 bp.