characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-10-31T17:01:50Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104041907
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-10-31T17:01:50Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1705800
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5732:CTCF_NS_rep2_CTCF
characteristics.characteristic: Submitter Id
characteristics.text: Millipore 07-729
characteristics.characteristic: antibody
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: 46C
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005484
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell derived neuronal stem cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: ES cells were plated at a density of 0.8 million cells per gelatin-coated 10 cm culture vessels in 50% DMEM, 50% F12 (DMEM/F12, Invitrogen) medium supplemented with 2.5ml of N2 and 5ml of B27 (Gibco), BSA (Gibco, final concentration of 0.012%), non-essential amino acids (Gibco), glucose (final concentration 0.03 M), HEPES (final concentration 4.5 mM) and 0.1 mM beta-mercaptoethanol (differentiation medium). Medium was exchanged after 24 and 48 hours, and the cultures were grown for an additional 72 hours. Cells were then gently dissociated using Accutase (Sigma), the GFP+ cell fraction (corresponding to ca. 70% of cells) was sorted by flow cytometry and seeded into a laminin (Sigma) coated 75 cm2 flask (final density of laminin: 10 µg/cm2 of culture surface, coating time: minimum 4 hours at 37°C). Subsequently, cells were grown in differentiation medium supplemented with in-house prepared recombinant murine EGF and bFGF (final concentration of 10 ng/ml) until loss of GFP expression and uniform upregulation of Nestin expression was observed by immunostaining and qRT-PCR. NS cells were passaged at 80% confluence. Medium was exchanged daily. Cells were crosslinked with 1% formaldehyde for 10min at RT, glycine was added to a final concentration of 0.125M to quench formaldehyde. Cells were lysed with 1 ml of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitors) for 20 minutes on ice. Chromatin was sonicated to average fragment size of 400bp. Chromatin extracts corresponding to 10M of cells (90µg of DNA) were precleared with 40µl of Protein A dynabeads. The sample was incubated at 4°C with overhead mixing for 1 hour. 40µl of protein A Dynabeads was washed with PBS and incubated with anti-CTCF (Milipore, 07-729, 5µl) or anti-Rad21 (Abcam, ab-992, 5µg) at RT with overhead mixing (40µl of beads per 5µl of anti-CTCF or 5µg of anti Rad21). 500µl of pre-cleared chromatin was mixed with the antibody-coupled beads and incubated overnight at 4°C with overhead mixing. Next, beads were washed for 10 minutes at 4°C with overhead mixing with lysis buffer (2x), lysis buffer containing 0.3M NaCl (2x), LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate, 2x), TE (pH 8.0) plus 0.2% Triton X-100, and TE (pH 8.0). Beads were resuspended in 100µl of TE (pH 8) and incubated at 65°C for 14 hours, 1µl of RNAseA was added and the sample incubated for 1 hour at 37°C. Next,10µl of Proteinase K was added and the sample incubated at 55°C for 2 hours. Beads were separated from the solution using a magnet. 110µl of the supernatant was mixed with 200µl of Ampure Beads (Beckman Coulter) and DNA was extracted following manufacturer’s suggestions. The Library was prepared in Ovation SP Ultralow library system (Nugen) using 2ng of ChIP material.
characteristics.characteristic: description
characteristics.text: N2B27
characteristics.characteristic: growth condition
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: naive embryonic stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: Biological_replicate2
characteristics.characteristic: replicate
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: CTCF_NS_rep2_CTCF
characteristics.characteristic: title