Illumina HiSeq 2500 sequencing; ChIP-Seq to investigate the opposing role of Dpf2 and Eed on differentiation of Mouse Embryonic Stem Cells via regulating Tbx3
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
2018-06-30
ENA last update
2017-11-01
ENA-CHECKLIST
ERC000011
External Id
SAMEA104364726
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-06-30T17:01:47Z
INSDC last update
2017-11-01T10:11:28Z
INSDC status
public
Submitter Id
E-MTAB-6165:Oct4_ChIP_OHT
broker name
ArrayExpress
cell line
E14-ES
cell type
embryonic stem cell
common name
house mouse
disease
normal
genotype
Dpf2 flox/flox
organism part
inner cell mass
sample name
E-MTAB-6165:Oct4_ChIP_OHT
Sequenced DNA Library
library_name
Input_OHT_rep1_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. To stabilize Dpf2 on chromatin, cells were treated with 2 mM disuccinimidyl glutarate (DSG) for 10 minutes prior to formaldehyde crosslinking. For all other targets, cells were chemically cross-linked at room temperature by the addition of formaldehyde to 1% final concentration for 10 minutes and quenched with 0.125 M final concentration of glycine. Cross-linked cells were re-suspended in sonication buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonicated using a Diagenode Bioruptor for three 10-minute rounds using pulsing settings (30 sec ON; 1 min OFF). 10 µg of sonicated chromatin was then incubated overnight at 4ºC with 5 µg of Flag antibody conjugated to magnetic beads. Following the IP, beads were washed twice with RIPA buffer (50mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% Na-deocycholate, 0.1% SDS), low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% Na-deoxycholate, 1% NP-40), and 1X TE. Finally, DNA was extracted by reverse crosslinking at 65ºC overnight with proteinase K (20ug/µL) and 1% SDS followed by phenol:chloroform:isoamyl alcohol purification and ethanol precipitation. Libraries were constructed as indicated above and sequenced using Illumina HiSeq 2500. The Dpf2 targeting vector was linearized and electroporated into R26::CreERT2 E14 cells to generate heterozygous ES cell lines after G418 selection. The selected heterozygous ES lines were transiently transfected with a FLP recombinase encoding plasmid (pCAGGsFlpE), converting the initial knockout allele (Dpf2+/-, lacZ positive, G418 resistant) into a “wild-type” (WT) allele with two floxP sites flanking exon 4 (Dpf2+/+, reverted WT (rWT), lacZ negative, G418 sensitive). Multiple independent rWT ES clones were then electroporated with the original Dpf2 knockout vector or Dpf2 critical exon-deleted targeting vector and selected with G418. Dpf2 critical-exon-deleted vector was generated by transforming Dpf2 targeting vector into the cre-containing bacteria. In the second round targeting, the clones recovered were only those with the second WT allele being targeted, which were confirmed by the presence of both the rWT allele and the second knockout allele. The long-range PCR reactions were carried out for genotyping (SequalPrep Long PCR kit, Invitrogen). The selected heterogeneous ES cell lines were converted to the conditional Dpf2+/+ and Dpf2+/- clones by transiently transfected with FlpE. ChIPed DNA was purified using Qiagen MinElute PCR Purification Kit. Library DNA was purified using AmpureXP beads using 1:1 ratio. Libraries were constructed using Diagenode MicroPlex ChIP kit.