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| Status |
Public on May 30, 2013 |
| Title |
PGC_H3K27me3_ChIPSeq |
| Sample type |
SRA |
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| Source name |
E11.5 Primordial Germ Cells, H3K27me3 ChIP
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| Organism |
Mus musculus |
| Characteristics |
strain/background: transgenic Oct4:GFP B6 crossed to Swiss-Webster
cell type: primordial germ cells (PGCs)
age: E11.5
chip antibody: anti-H3K27me3
library amplification cycles: 20
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| Growth protocol |
We used a transgenic Oct4:GFP B6 mouse line crossed to Swiss-Webster females. The gonadal regions from multiple embryos were isolated at E11.5 and pooled prior to enzymatic dissociation. Primordial germ cells (PGCs) were isolated by FACS as the GFP+ population.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Two aliquots of E11.5 PGCs, consisting of 104,000 and 97,000 cells, were cross-linked in 0.25% formaldehyde (Thermo Scientific 28906) in PBS for 10min at room temperature prior to quenching with 125mM glycine. Cross-linked material was sonicated using a Covaris sonicator for 12' at Duty 5%, Intensity 3, and Bursts 200. Lysate for each replicate was diluted in lysis buffer and cleared of debris. Cleared lysate from each replicate was divided equally to perform ChIP for H3K4me3, H3K27me3, and an input control. For each IP, 11µl of protein A Dynabeads (Life Technologies 1001D) were pre-incubated with 2.4µg of either anti H3K4me3 (Diagenode pAb003-050, lot # A5051-001P) or H3K27me3 (Millipore 07-449, lot # 1999681) in ChIP lysis buffer. Lysates were incubated with preformed bead/antibody overnight at 4C with mixing. The chromatin/bead/antibody complexes were washed sequentially with lysis buffer three times, DOC buffer once, and TE buffer once, followed by a transfer to a fresh PCR tube. Chromatin was eluted using elution buffer (1% SDS, 0.1M NaHCO3). IP and Input samples were treated with RNaseA followed by Proteinase K treatment. Cross-linking was reversed by incubating overnight at 65C while shaking. IP-DNA and Inputs were purified using a MinElute PCR Purification Kit (Qiagen 28004).
Libraries were generated using the ThruPLEX-FD Prep Kit (Rubicon Genomics R40048) with 20 cycles of amplification for IP-DNA and 15 cycles for Input DNA.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Chromatin IP against H3K27me3 (Millipore 07-449. lot # 1999681)
This Sample represents 2 replicates.
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| Data processing |
Reads from each ChIP-Seq library were filtered to retain only unique sequences. Fiitered reads were aligned to the mm9/NCBI build 37 mouse genome using bowtie (v0.12.7) and reads mapping only once were retained.
A 13-state segmentation of the genome was generated using ChromHMM (v1.06) to call and compare H3K4me3- and H3K27me3-enriched regions in PGCs and ESCs (ESC data obtained from GEO accession GSE12241 and handled in the same manner as PGC data). The four samples (PGC H3K4me3, PGC H3K27me3, ESC H3K4me3, ESC H3K27me3) were treated as distinct marks from a single cell type to allow a direct and unbiased comparison of the genome segmentation in PGCs and ESCs. K-means clustering was used to group the state emission parameters for each sample into 2 groups ("on" or "off"), and these were used to classify each of the 13 ChromHMM segmentation states as "bivalent", "H3K4me3 only", "H3K27me3 only", or "none" for each cell type. Data from the replicate PGC libraries were pooled together for this analysis. H3 ChIP-Seq data from the ESC dataset (GEO accession GSE12241) was used as control for the ESC samples.
All genomic segments from the ChromHMM PGC H3K4me3- or PGC H3K27me3-containing ("on") states were merged into summary bed files.
Genome_build: MGSCv37
Supplementary_files_format_and_content: All files in UCSC BED format.
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| Submission date |
Apr 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Courtney Onodera |
| E-mail(s) |
courtney.onodera@ucsf.edu
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| Organization name |
University of California, San Francisco
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| Street address |
1450 Third St, HD374
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE46396 |
Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline in Vivo |
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| Relations |
| BioSample |
SAMN02056367 |
| SRA |
SRX272026 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1129745_PGC_Sachs_H3K27me3_chromHMM_contiguous.bed.gz |
171.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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