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| Status |
Public on Feb 17, 2014 |
| Title |
WT_H3K27me3_ChIPSeq |
| Sample type |
SRA |
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| Source name |
spleen, WT, H3K27me3 ChIP
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| Organism |
Mus musculus |
| Characteristics |
strain/background: 129Sv
genotype: RBP-J +/+;Ren1dcre/+
age: 261 days
tumors: no
tissue: spleen
chip antibody: H3K27me3 (Millipore Cat.#07-449 Lot. JBC1854858)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Spleens were removed from the mice, snap frozen in liquid N2 and stored at -80°C until shipment on dry ice to ActiveMotif. For fixation, spleens were cut into small pieces in PBS + 1% formaldehyde and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycin (final) and tissue pieces were treated with a TissueTearer. Chromatin was isolated by disrupting the cells with a Dounce homogenizer. Lysates were sonicated using a Misonix Sonicator 3000 equipped with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and stored at -80 C. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNaseA, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer. For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using precleared chromatin and antibodies and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. Immune complexes were eluted from the beads with SDS buffer, and subjected to RNase treatment and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3'-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (250-350 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on HiSeq 2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Sequences: Standard Illumina software base-calling and quality-control filtering was applied.
Sequences (50 nt reads, single end) were aligned to the mouse genome (mm9) using the BWA algorithm (default parameters).
Aligns were extended in silico at their 3'-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic "signal maps") were stored in BAR files.
Peak locations were determined using the SICER algorithm (v1.1) with a cutoff of FDR = 1E-10 and a Gap size of 3x fragment size (200 bp) for H3K27me3 and 1x fragment size for H3K4me3.
Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations.
Genome_build: MGSCv37
Supplementary_files_format_and_content: BAR = Signal map of fragment densities in binary format (Affymetrix BAR format).
Supplementary_files_format_and_content: BEDGRAPH = Standard bedGraph file containing SICER islands (peak regions).
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| Submission date |
May 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
R. Ariel Gomez |
| E-mail(s) |
rg@virginia.edu
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| Phone |
434-924-2525
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| Fax |
434-982-4328
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| Organization name |
University of Virginia
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| Department |
Pediatrics
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| Lab |
Gomez
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| Street address |
409 Lane Rd. MR-4 2001
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| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE46566 |
Mouse spleen: Histone modification patterns in spleens from wild type and leukemic mice |
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| Relations |
| BioSample |
SAMN02116179 |
| SRA |
SRX273302 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1132536_1_WT_H3K27me3_i12_uniqnorm_hits-W200-G600-FDR1E-10-island.bedGraph.gz |
202.7 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM1132536_1_WT_H3K27me3_i12_uniqnorm_signal.bar.gz |
98.7 Mb |
(ftp)(http) |
BAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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