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| Status |
Public on Mar 09, 2012 |
| Title |
Reverb_alpha_null_5pm |
| Sample type |
SRA |
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| Source name |
Homogenized mouse liver, Rev-erb(alpha) KO, 5pm/ZT10, Rev-erb(alpha) ChIP
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: Rev-erb(alpha) KO
gender: male
age: 8-12 weeks
tissue: liver
time: 5pm/ZT10
chip antibody: Rev-erb(alpha)
antibody vendor: Cell Signalling
antibody catalog #: 2124
antibody lot#: 2
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| Treatment protocol |
Liver was harvested immediately after euthanasia, quickly minced and cross-linked in 1% formaldehyde for 20min, followed by quenching with glycine solution. Nuclear extracts were prepared by Dounce homogenization. Chromatin fragmentation was performed by sonication using the Bioruptor (Diagenode). Proteins were immunoprecipited using Rev-erbβ antiserum or Rev-erbα antibody (Cell Signalling #2124). Cross-linking was reversed overnight at 65°C, and DNA was isolated using phenol/chloroform/isoamyl alcohol.
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| Growth protocol |
Male C57BL/6 WT and Rev-erbα KO mice, 8-12 weeks of age, were housed under 12h-light/12h-dark cycles (lights on at 7 AM, lights off at 7 PM), and euthanized at ZT10 (5PM) or ZT22 (5AM).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed independently on liver samples from 4 different mice and subsequently pooled. Libraries for each ChIP-Seq experiment were prepared as per Illumina's instructions (http://www.illumina.com). Briefly, DNA fragments were blunted, phosphorylated, and ligated to Illumina library adapters. Size selection was performed using gel electrophoresis by excising DNA fragments at 200 ± 50 base pairs. Following gel purification, PCR amplification was performed (30 s at 98°C; [10 s at 98°C, 30 s at 65°C, 30 s at 72°C] × 18 cycles; 5 min at 72°C). Amplified material was evaluated on an Agilent 2100 bioanalyzer to ensure proper size selection.
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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 |
Mapped read BED files: For all ChIP-Seq samples, sequence reads were mapped to the mouse genome (NCBI36/UCSC mm8) using ELAND software. Only the sequences uniquely mapped with no more than 2 mismatches in the first 32 bp were kept and used as valid reads. Redundant reads mapping to the same genomic loci were condensed to a single read to remove clonal amplification artifacts. Reads from replicate samples were pooled together prior to further analysis
Peak Call BED files: Peak calling was carried out by HOMER (version 3, 0.1% FDR, 1rpm cutoff).
Analysis BED files: To adjust for the higher read depth in the Rev-erb(alpha)-null ChIP-Seq sample, reads were randomly sampled from the Rev-erb(alpha)-null data 10 times, such that each random sample had a similar read count to the wild-type cistrome. The peak height was computed in each random sample, and the average height at each peak was used for subsequent analysis. In order to obtain the most stringent high confidence cistrome, potential non-specific binding events in the Rev-erb(alpha) 5PM/ZT10 cistrome was identified, thereby removing 5,830 peaks where the Rev-erb(alpha) ChIP-Seq profile in the Rev-erb(alpha)-null animal was more than 50% of the peak height observed in the WT at 5PM/ZT10 from all subsequent analysis. The remaining peak calls for both Rev-erb(alpha) and Rev-erb(beta) were then merged together, such that any overlapping regions were combined into one region covering the peak calls for both isoforms. This resulted in 31,958 total Rev-erb binding regions, which contain a valid peak call for Rev-erb(alpha) and/or Rev-erb(beta). The ChIP-Seq signal for each subtype was then quantified at each region by computing the maximum stack height profile within the region, normalized to the total number of reads for that isoform (rpm). Each region was categorized as being specific to either subtype, or common to both isoforms, as follows: First, Fisher’s Exact Test was applied to each region, comparing the peak height and total reads for each factor for significant changes that are unlikely to occur simply due to variability in the sequencing process. Regions with a Benjamini-Hochberg corrected p-value <= 0.05 from Fisher's Exact Test, and an absolute fold-change > 2 using the normalized (rpm) peak heights, were considered to be specific to the subtype with greater binding. All other sites were considered “common” sites because, regardless of peak calls, these sites did not show a significant quantitative difference in binding signal between the two subtypes. By this method, 29,462 sites are considered “common” sites (92%), 1,614 are considered "Alpha-specific" (5%), and 882 are considered "Beta-specific" (3%).
Genome Build:
ReverbA_KO_Liver_ZT10_masked.bed: mm8
reverb_alpha_KO_zt10_r1_223_2_rd.bed.gz: mm8
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| Submission date |
Mar 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Logan J Everett |
| Organization name |
U.S. Environmental Protection Agency
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| Department |
Office of Research and Development
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| Lab |
Center for Computational Toxicology and Exposure
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| Street address |
109 T.W. Alexander Dr
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| City |
RTP |
| State/province |
North Carolina |
| ZIP/Postal code |
27711 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE36375 |
Rev-erb(alpha) and (beta) Coordinately Protect the Circadian Clock and Normal Metabolic Function |
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| Relations |
| SRA |
SRX128176 |
| BioSample |
SAMN00809652 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM890127_ReverbA_KO_Liver_ZT10_masked.bed.gz |
80.5 Kb |
(ftp)(http) |
BED |
| GSM890127_reverb_alpha_KO_zt10_r1_223_2_rd.bed.gz |
1.0 Gb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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