Illumina HiSeq 3000 sequencing; Gain of chromatin loops marks the exit from naive pluripotency - ChIP seq for CTCF and Rad21
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Rad21
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
ENA first public
2017-10-31
ENA last update
2017-05-16
ENA-CHECKLIST
ERC000011
External Id
SAMEA104041908
INSDC center alias
unspecified
INSDC center name
unspecified
INSDC first public
2017-10-31T17:01:50Z
INSDC last update
2017-05-16T18:32:04Z
INSDC status
public
Submitter Id
E-MTAB-5732:Rad21_NS_rep1_Rad21
antibody
abcam ab992
broker name
ArrayExpress
cell line
46C
cell type
embryonic stem cell derived neuronal stem cell
common name
house mouse
growth condition
N2B27
progenitor cell type
naive embryonic stem cell
replicate
Biological_replicate1
sample name
E-MTAB-5732:Rad21_NS_rep1_Rad21
Sequenced DNA Library
library_name
Rad21_NS_rep1_Rad21_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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.