Illumina Genome Analyzer II sequencing; Analysis of binding of the transcriptional factor Nrf1 in mouse embryonic stem cells
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
ENA checklist
ERC000011
INSDC center alias
GIS
INSDC center name
Genome Institute of Singapore
INSDC first public
2017-12-15T17:02:24Z
INSDC last update
2018-03-09T00:15:49Z
INSDC status
public
SRA accession
ERS699903
Sample Name
ERS699903
alias
E-MTAB-3475:Nrf1
broker name
ArrayExpress
cell line
ES-E14 cell
organism
Mus musculus
title
Nrf1
Sequenced DNA Library
library_name
Nrf1
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Mouse E14 and Oct4-Gip ESCs were plated on 0,1% gelatin coated plates (Sigma G1393) and maintained in DMEM (Invitrogen 11960-044) supplemented with either 15% KSR (Invitrogen 10828-028) or 15% FBS (Hyclone), 1mM L-glutamine (Invitrogen 25030/081), 0,1 mM of non-essential aminoacids (Invitrogen 11140-050), 100 ug/ml penicillin/streptomycin (Invitrogen 15140-122), 1 mM sodium pyruvate (Invitrogen 11360-070), 0,1 mM mercaptoethanol (Sigma M7522), and Esgro 103U/ml (Chemicon ESG1106). Cells were passaged upon reaching 80% confluence. Growth media was aspirated and cells were washed using warm PBS. Cells were dissociated by incubating them with trypsin/EDTA (Invitrogen 15090-046) for 3 to 5 minutes. Once most of the cells are observed to be at a single cell dissociation state, they were collected and spun down for 5 minutes at 1200 rpms. After pellet ressuspention cells were plated in a gelatin coated plated at an average density of 21000 cells per cm2 . Embryoid bodies were formed by plating ES cells in non-attachment conditions (suspension culture) with ES cell medium with fetal bovine serum and in the absence of Esgro, for 21 days. ChIP-qPCR was performed as described by Upstate Biotechnology with minor adaptations: cells were first crosslinked with 1.5mM EGS (proteochem-c1130-100mg) per 10 ml of iced PBS, cells were incubated on a nutator for 45 minutes. After 3 PBS washes cells are crosslinked with 1% formaldehyde for 10 mins with soft orbital agitation. Formaldehyde is quenched with 250 mM of glycine for 5 minutes and triple washed with PBS. Cells were sonicated post lysis in a bioruptor for 40 min total (7 sec on/15 sec off on high). Immunoprecipitation was achieved using an anti-NRF1 antibody (ab34682) and with an IgG control (ab46540). DNA was extracted using qiagen PCR purification kit.