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Status |
Public on Sep 10, 2015 |
Title |
MC1-ZE7 cells (Em+ high fraction) H3K27ac rep2 |
Sample type |
SRA |
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Source name |
MC1-ZE7 ES cells
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Organism |
Mus musculus |
Characteristics |
cell type: FACS sorted Emerald+ ES cells strain: MC1 (129S6/SvEvTac) chip antibody: H3K27ac (Active Motif #39133)
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Treatment protocol |
The ES cells were FACS-sorted according to the fluorescent intensity of Emerald.
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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 |
FACS sorted MC1-ZE7 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped by 125 mM glycin. The cells were washed with PBS and stored at –80°C prior to use. The cells were lysed in Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) containing proteinase inhibitor cocktail (Roche). Sonication was coducted with the Misonix sonicator XL to generate DNA fragments of approximately 150-450 bp. The sonicated lysates were diluted in ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) containing proteinase inhibitor cocktail and incubated overnight at 4°C with 30 µl of protein G magnetic beads (Invitrogen) that had been preincubated for 3 h with 3 µg of antibodies. The precipitants were washed three times with RIPA buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and once with 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10 mM NaCl. Bound chromatin was eluted from the beads at 70°C, 1,300 r.p.m in Elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS), followed by crosslink reversal at 70°C for 6 h. After treatment with RNase A and proteinase K, DNA was purified by a phenol-chloroform-isoamylalcohol extraction and isopropanol precipitation. Library preparation was performed using ChIP-seq sample preparation kit (Illumina) with several modifications as follows. A 1:30 dilution of the Adaptor Oligo Mix was used in the ligation step. A range of fragment size (200-300 bp) was selected by separation on a 2% low melting temperature agarose gel (Lonza). DNA purification in each reaction was carried out using AMPure XP beads (Beckman). In the experiment of second replication, PCR amplification were coducted prior to size selection and a range of fragment size (200-500 bp) was selected. Sequencing analysis was performed on the Illumina Genome Analyzer II according to the manufacturer’s instructions.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer IIx |
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Description |
H3K27ac Zscan4+ rep2
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Data processing |
ChIP-seq reads were aligned to the mouse genome (mm9) using Bowtie with maximum 2 mismatches allowed and maximum number of matches in the genome <=10 (so that duplicated regions in the genome, such as Zscan4 gene cluster, are still covered by ChIP-seq reads). Redundant reads (that start from the same nucleotide) were removed. To compare genome-wide histone H3K27 acetylation we counted the number of tags per each 1 Kb interval in each replication of Zscna4+ and Zscan4- cells. Intervals that had <15 total reads in all were combined with neighboring genome intervals. Then, we used ANOVA with error variance adjustment (maximum of actual error variance and average error variance for genome segments with a similar number of total reads) using the algorithm of NIA Array Analysis6. The difference was considered statistically significant based on FDR <0.05, to account for multiple comparison tests. Genome regions with differential histone acetylation were associated with the genes based on the distance <100 Kb from the transcription start site (TSS). Genes located at distances >5 fold the minimal distance were not analyzed. To identify peaks of H3K27ac we used HOMER findPeaks (http://biowhat.ucsd.edu/homer/ngs/index.html); peaks identified using "factor" and "histone" modes were combined for each ChIP-seq sample. Peaks wider than 2 Kb were not analyzed because they did not accurately predict genome locations. To select reproducible peaks, we selected for analysis only those peks that overlapped in both ChIP-seq replications either in Zscan4+ or Zscan4- cells. Peaks identified in Zscan4+ or Zscan4- cells were combined if they overlapped or were separated by <500 bp. Then ChIP-seq reads were counted in each peak, normalized by the total number of mapped non-redundatn sequence reads. The logratio of Em+/Em- was estimated for each peak, and then was used to sort the peaks. Genome_build: mm9 Supplementary_files_format_and_content: peak files
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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) |
GSE51679 |
ChIP-seq assays for H3K27ac |
GSE51682 |
Zscan4 mediates transient remodeling and transcriptional burst of heterochromatin in mouse embryonic stem cells |
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Relations |
BioSample |
SAMN02383596 |
SRA |
SRX367523 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1250436_Z+_rep2_RPKM.txt.gz |
438.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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