Neural stem cell culturing and derivation: Wild-type NSCs were cultured using N2B27 medium (Stem Cell Sciences) supplemented with EGF and FGF (both from Peprotech). For large scale chromatin preparation, 108 NSCs were crosslinked with 2 mM disuccinimidyl glutarate (Thermo Fisher Scientific) and 1% formaldehyde, nuclei were isolated in 50 mM Tris-Cl pH 8.0, 1 mM EDTA, 1%SDS, lysed in pre-IP buffer (10 mM Tris, 10 mM NaCl, 3mM MgCl2, 1 mM CaCl2). Chromatin was prepared and ChIP performed according to the Millipore on-line protocol using 15 mg of antibody against Chd7 antibody (ab-31824, Abcam).
INSDC center name
BCCS
INSDC first public
2011-03-14T14:25:27Z
INSDC last update
2013-11-04T20:04:46Z
INSDC status
public
SRA accession
ERS017933
Sample Name
ERS017933
Title
Mus musculus
Sequenced DNA Library
library_name
Chd7 ChIP
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP DNA is prepared for analysis on the Illumina Cluster Station and Genome Analyzer according to the Illumina protocol (www.illumina.com). Briefly, 10 ng of ChIP DNA is end-repaired and purified through a QiaQuick PCR purification column (Qiagen). Blunt ended fragments are A-tailed using Klenow exo-enzyme in the presence of dATP, purified using a MinElute PCR purification column (Qiagen) and suspended in 10 μl of elution buffer. Illumina-provided single read adapters are ligated to the A-tailed DNA fragments, and ligated products are run on a 2% agarose gel. DNA fragments are size-selected on gel by excising gel slice of the 200 ± 25 bp range. DNA is eluted in 36 μl of elution buffer after purification on a Qiagen Gel extraction column. The resulting adapter-modified DNA fragments are enriched by PCR using Phusion polymerase as follow: 30 s at 98 °C, 18 cycles of (10 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C), 5 min at 72 °C final extension. PCR products are purified using MinElute PCR purification columns and eluted in 15 μl of elution buffer. One microliter is loaded on an Agilent Technologies 2100 Bioanalyzer using a DNA 1000 assay to determine the library concentration and to check for quality.