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Sample GSM1004653 Query DataSets for GSM1004653
Status Public on Oct 21, 2012
Title ESC merged mononucleosomes
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics strain: 129P2/Ola
cell type: mouse embryonic stem cells
passage: 15-25
developmental stage: blastocyst, E3.5
Extracted molecule genomic DNA
Extraction protocol 129P2/Ola embryonic stem cells were cultured in ESGRO Complete (Millipore). Differentiation of ESC into neuronal precursors was induced via formation of embryoid bodies in embryoid body formation medium (Millipore) and treatment with 5 µM retinoic acid for four days. Neuronal embryoid bodies were dissociated and seeded on matrigel in neuronal stem cell medium (PAN) for four days. 129P2/Ola mouse embryonic fibroblasts (MEFs) were generated from pregnant E13.5 mice and cultured in DMEM supplemented with 10% fetal calf serum and glutamine up to passage five. For MNase digestion, cells were harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 °C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM Tris-HCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 Units MNase/µl (Fermentas) and incubation for 6-11 minutes at 37°C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 µg/µl RNase A (Fermentas) and removal of protein by phenol/chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2% agarose gel using an E-Gel electrophoresis system (Life Technologies, Carlsbad, CA, USA). The libraries for sequencing were prepared according to the standard protocol for the Illumina HiSeq2000 sequencing platform.
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina HiSeq 2000
Data processing DNA reads were aligned on the mm9 assembly version of the mouse genome with Bowtie reporting unique hits with up to two mismatches.
The nucleosome occupancy maps were calculated with custom made Perl scripts by counting how many paired-end reads covered a given DNA base pair.
Sites with artificially high coverage were considered as artifacts and excluded from the analysis.
No further peak calling or smoothing was conducted. In addition, no assumptions on the length of the nucleosomal DNA had to be made to derive the nucleosome occupancy maps, since nucleosome boundaries were determined on both sides of the nucleosome.
Genome_build: mm9
Supplementary_files_format_and_content: Nucleosome Nucleosome positioning data are in tab separated text files with four columns: (1) Chromosome #, (2) nucleosome start site, (3) nucleosome end site, (4) MNase-protected fragment length.
Submission date Sep 14, 2012
Last update date May 15, 2019
Contact name Vladimir B Teif
Organization name University of Essex
Street address Wivenhoe Park
City Colchester
ZIP/Postal code CO4 3SQ
Country United Kingdom
Platform ID GPL13112
Series (2)
GSE40896 Genome-wide nucleosome positioning during embryonic stem cell development
GSE40910 Genome-wide nucleosome positioning during embryonic stem cell development [MNase-Seq]
SRA SRX187610
BioSample SAMN01178487

Supplementary file Size Download File type/resource
GSM1004653_ESCmono_merged_nucleosomes.bed.gz 3.9 Gb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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