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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 20, 2013 |
| Title |
Luminal K4me3 ChIPSeq |
| Sample type |
SRA |
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| Source name |
mammary gland
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| Organism |
Mus musculus |
| Characteristics |
cell type: Luminal cells
state: virgin
chip antibody: Histone H3 trimethyl Lys4
strain: FVB/N
chip antibody manufacturer: Millipore
chip antibody catalog #: 07-473
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| Treatment protocol |
Cells obtained by Flow cytometry were fixed with paraformaldehyde and snap frozen
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| Growth protocol |
Freshly sorted cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The standard ChIP protocol was used to isolate genomic DNA marked with the above histone modifications.
Libraries for paired-end sequencing were prepared by Geneworks using 10 ng ChIP DNA according to the Illumina ChIP-seq Sample Preparation protocol, revision A, with the following modifications. Paired-end adapters were substituted for standard genomic adapters, and library amplification (18 cycles) performed using primers PE 1.0 and 2.0. Size selection for 300 bp DNA was performed prior to amplification. Final libraries were analyzed using the Agilent Bioanalyzer High Sensitivity DNA Kit and sequenced on the Illumina Genome Analyzer IIx running SCS 2.8, generating 35 base paired-end reads using the CASAVA 1.7 analysis pipeline.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalls performed using CASAVA version 1.7
Reads were mapped to the mouse reference genome mm9 using the Subread aligner (http://subread.sourceforge.net). Six subreads were selected from each read and at least two consensus subreads were required for reporting a hit. No mismatches were allowed when mapping extracted subreads to the reference genome. A fragment was judged to be successfully mapped if the paired ends mapped to locations between 50 and 500 bp apart.
Mapped fragments were assigned to each gene by comparing the location of fragments to the chromosomal coordinates of regions defined for each gene. For H3K27me3 marks, each gene was assigned two values in each sample, which gave the H3K37me3 marking levels for the TSS region and the broad region. For H3K4me3 marks, only marking levels in the TSS region were used. The ‘TSS’ region is defined as the region 3 kb upstream and 2 kb downstream sequence of the transcriptional start site of the gene, and the ‘broad’ region defined as the genomic span of the gene including 3kb of sequence upstream of the transcriptional start site.
Statistical analysis of the count data was performed using edgeR package of the Bioconductor software project. Genes were called as significantly enriched for each histone mark using the normalizeChIPtoInput function, which normalizes the ChIP counts to input and evaluates enrichment using a negative binomial statistical model. Log2-fold-changes in histone mark coverage between cell populations or between conditions were computed using the predFC function. This function adjusted the ChIP counts for input by fitting a negative binomial log-linear generalized linear model, using a prior count of 0.5 per sample to avoid unreliably large log-fold-changes that might otherwise arise from zero or small counts. The negative binomial dispersion was set to 0.01 for all calculations, allowing for a degree of biological variation typical of mouse experiments.
Genome_build: mm9
Supplementary_files_format_and_content: Bed fies were generated using in-house C code. They include the chromosomal coordinates of each mapped fragment.
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| Submission date |
Dec 31, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Wei Shi |
| E-mail(s) |
Wei.Shi@onjcri.org.au
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| Organization name |
Olivia Newton-John Cancer Research Institute
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| Street address |
145 Studley Road
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| City |
Heidelberg |
| State/province |
Victoria |
| ZIP/Postal code |
3084 |
| Country |
Australia |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE43212 |
Histone methylation profiles of prospectively isolated mammary epithelial subpopulations |
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| Relations |
| SRA |
SRX213412 |
| BioSample |
SAMN01881590 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1058735_HSA_Vrgn_K4.bed.gz |
243.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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