alias: E-MTAB-3475:Nrf1
title: Illumina Genome Analyzer II sequencing; Analysis of binding of the transcriptional factor Nrf1 in mouse embryonic stem cells
design: Analysis of binding of the transcriptional factor Nrf1 in mouse embryonic stem cells
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.
identifiers.namespace: GIS
identifiers.id: E-MTAB-3475:Nrf1
identifiers.namespace: Genome Institute of Singapore
identifiers.id: E-MTAB-3475:Nrf1