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| Status |
Public on May 03, 2017 |
| Title |
H3K27me3-AB1-Adult-Rod_I_N-P3 replicate 1 |
| Sample type |
SRA |
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| Source name |
retina
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: postnatal day 3
chip antibody: H3K27me3(Millipore,07-449)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP with iDeal ChIP-seq kit (Diagenode # C01010051), MinElute PCR-purification kit (QIAGEN #28006), quantified using the Quant-iT PicoGreen ds DNA assay (Life Technologies #Q33120)
Libraries were prepared from 5-10 ng DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext High-Fidelity 2x PCR Master Mix according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA) with the following modifications: A 1:1 Ampure cleanup was added after adaptor ligation a total of 2 times.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Adult-Rod_I_N-P3-H3K27me3.bw
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| Data processing |
Basecalls performed using CASAVA
BWA (version 0.5.9-r26-dev, default parameter) to align the ChIP-Seq reads to mouse genome mm9(MGSCv37 from Sanger), Picard(version 1.65(1160)) then have been used for marking duplicated reads. Then only non-duplicated reads with have been kept by samtools (parameter “-q 1 -F 1024” version 0.1.18 (r982:295)). We followed the ENCODE criterion to quality control (QC) the data that non-duplicated version of SPP (version 1.11) have been used to draw cross-correlation and calculated relative strand correlation value (RSC) under support of R (version 2.14.0) with packages caTools(version 1.17) and bitops(version 1.0-6) and estimated the fragment size.
We required > 10M unique mapped reads for point-source factor (H3K4me2/3, H3K9/14Ac, H3K27Ac, CTCF, RNAPolII, BRD4) and RSC > 1. We required 20M unique mapped reads for broad markers (H3K9me3, H3K27me3, H3K36me3). We required 10M unique mapped reads for INPUTs and RSC < 1. We noticed H3K4me1 is point-source factor in some stages while broad in other stages so we QC H3K4me1 as broad markers. Then upon manually inspection, the cross-correlation plot generated by SPP, the best fragment size estimated (the smallest fragment size estimated by SPP in all our cases) were used to extend each reads and generate bigwig file to view on IGV (version 2.3.40).
All profiles were manually inspected for clear peaks and good signal to noise separation. For mouse data, all data broad markers with RSC < 0.8 have biological replicates except several samples with one of antibody for H3K9me3(due to availability of outsourcing), we think the quality of those H3K9me3 are good since they show clear peaks and RSC are bigger than most of the H3K9me3 data available in ENCODE or Epigenomics Roadmap.
To estimate the consistence between biological replicates, for point-source factors, MACS2 (version 2.0.9 20111102, option “nomodel” with “extsize” defined as fragment size estimated above) have been used to call peaks with FDR corrected p-value cutoff 0.05(strong peak set) and 0.5(weak peak set) separately, peaks within 100bp have been merged by bedtools (version 2.17.0). We required > 80% of strong peak set from either one replicate overlapping the weak peak set from the other replicate (median percentage 96.8%). For broad peaks, SICER (version 1.1 redundancy threshold 1, window size 200bp, effective genome fraction 0.86, gap size 600bp, FDR 0.00001 with fragment size defined above) has been used for domain calling and we required > 70% of domains called overlapping the domains called from the other replicate(median percentage 89.9%). After confirmed the consistency between replicates, we pool extended reads and generate the final bigwig track for visualization.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: bigwig
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| Submission date |
Sep 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Beisi Xu |
| E-mail(s) |
beisi.xu@stjude.org
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| Organization name |
St Jude Children's Research Hosipital
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| Department |
Center for Applied Bioinformatics
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| Street address |
262 Danny Thomas Pl
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE87037 |
The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis [ChIP-Seq_Mm] |
| GSE87064 |
The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis |
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| Relations |
| BioSample |
SAMN05785106 |
| SRA |
SRX2172692 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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