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Status |
Public on Sep 10, 2015 |
Title |
MC1-ZE7 cells (Em+ high fraction) Hpa II |
Sample type |
SRA |
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Source name |
DNAme_Em+_HpaII
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Organism |
Mus musculus |
Characteristics |
cell type: FACS sorted Emerald+ ES cells strain: MC1 (129S6/SvEvTac) passages: p31
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Treatment protocol |
The ES cells were FACS-sorted according to the fluorescent intensity of Emerald and centrifuged to collect. The cells were snap-frozen and stored in -80 C.
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Growth protocol |
MC1-ZE7 ES cells were cultured on gelatin-coated feeder-free plates in complete ES medium, DMEM (Gibco), 15% FBS (Atlanta Biologicals), 1000 U/ml leukemia inhibitory factor (LIF) (ESGRO, Chemicon), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (NEAA), 2 mM GlutaMAX, 0.1 mM beta-mercaptoethanol, and penicillin/streptomycin (50 U/50 µg/ml). Medium was changed daily and cells were split every 2 to 3 days routinely.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was extracted from individual cell populations separately. Cells were lysed in sodium dodecyl sulfate (SDS)-containing DNA extraction buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 50 mM EDTA) and treated with proteinase K for 3 h at 55°C. The residual RNA was digested by incubation in the presence of RNase A for 1 h in 37°C. Lysates were subjected to three rounds of phenol–chloroform–isoamylalcohol (PCI; Invitrogen) extraction and the genomic DNA was precipitated by ethanol, washed with 70% ethanol, dried, and then dissolved in TE (10 mM Tris-HCl, pH 8.0, 1mM EDTA). The quality of purified genomic DNA was checked by gel and Nanodrop (260/280=1.7-1.9, 260/230>2). The HELP tagging assay was performed on purified genomic DNAs. The protocol was modified for NEBNext Multiplex Oligos for Illumina (NEB) from the original protocol (http://wasp.einstein.yu.edu/index.php/Protocol:HELP_tagging). Briefly, genomic DNA was digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. AS and AE adapters were prepared by annealing two oligo DNAs separately. The first Illumina AE adapter was ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking genomic DNA sequence. An A-overhang was created, allowing the ligation of the second Illumina AS adapter. This created both AE-insert-AS products and AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we selectively enriched for the AE-insert-AS product as single-strand RNA. After cDNA synthesis by specific primers, PCR amplification was performed corresponding primer set. The quantification was performed by KAPA quantification kit.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina Genome Analyzer IIx |
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Description |
pZscan4c-Emerald (Em+High) Hpa II library strategy: HELP tag sequencing
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Data processing |
HELP pipeline is described in Suzuki et al. Genome Biol 2010 (PMID: 20359321). To quantify DNA methylation in Em+ and Em- sorted cells we used data from HELP tagging method at genome locations within 1 kb from centers of each H3K27ac peak. DNA methylation was inferred from angle <50, where angle was estimated as: 100*atan(x/y)/(pi/2) 5. Peaks of H3K27ac, where <3 tags were obtained with HpaII restriction enzyme, were not used in the analysis. Decreased in DNA methylation at H3K27ac peaks was inferred from the increase of >=1.5 fold in the number of tags obtained with HpaII restriction enzyme. Peaks were sorted by the decreasing logratio of H3K27ac in Em+/Em- cells, and then the proportion of peaks with DNA methylation or decreased DNA methylation in Em+ vs. Em- cells was estimated in a sliding window of 300 peaks. Presence of chromatin modifications and cofactors was evaluated using ChIP-seq data. The number of ChIP-seq tags was then counted in each H3K27ac peak for Em+ and Em- cells. If the width of a peak was <1 kb, then ChIP-seq tags was then counted in a 1 kb intervals centered at the peak. If tag counts were >2 fold more abundant than in control, then the histone modification or cofactor was considered present. DNA methylation (>50%) was assessed using genome-wide bisulfite sequencing data9. Lamin B1 binding and abundance of H3K9me2 histone modifications was inferred from positive log-ratio of signal intensity in tiling array experiments10,11 for oligos aligned to each H3K27ac peak. Probes within 1 kb centered at H3K27ac peak were averaged. The proportion of H3K27ac peaks with histone modifications or cofactors was estimated separately in H3K27ac peaks that had increased acetylation (>3 fold) in Em+ cells compared to Em- cells, and in other H3K27ac peaks. Statistical significance was evaluated using a hypergeometric distribution as a null-hypothesis. A portion of these data (DNA methylation, lamin B1 binding, and H3K9me2 histone marks) was used to generate. Peaks were sorted by decreasing logratio of H3K27ac between Em+ and Em- cells, and then the proportion of peaks with >50% DNA methylation or average logratio for lamin B1 binding and H3K9me2 histone marks was estimated in a sliding window of 300 peaks. Genome_build: mm9 Supplementary_files_format_and_content: *hcount.txt file contents: Supplementary_files_format_and_content: (1) MspI site ID Supplementary_files_format_and_content: (2) Chromosome Supplementary_files_format_and_content: (3) MspI site position Supplementary_files_format_and_content: (4) Number of forward reads Supplementary_files_format_and_content: (5) Number of reverse reads Supplementary_files_format_and_content: (6) Total number of reads
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Submission date |
Oct 24, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Minoru S.H. Ko |
E-mail(s) |
kom@mail.nih.gov
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Phone |
410-558-8359
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Organization name |
NIH
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Department |
National Institute on Aging
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Lab |
Lab of Genetics
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Street address |
251 Bayview Blvd, Suite 100, 10C
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City |
Baltimore |
State/province |
MD |
ZIP/Postal code |
21224 |
Country |
USA |
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Platform ID |
GPL11002 |
Series (2) |
GSE51680 |
Genome-wide DNA methylation analyses by the HELP assay |
GSE51682 |
Zscan4 mediates transient remodeling and transcriptional burst of heterochromatin in mouse embryonic stem cells |
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Relations |
BioSample |
SAMN02383603 |
SRA |
SRX367524 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1250437_L1_hcount.txt.gz |
11.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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