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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 08, 2016 |
| Title |
MNase-seq in Adult Liver Low Rep 1.0 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
background strain: C3H
tissue: liver
developmental stage: adult
replicate: 1
mnase concentration: Low
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| Treatment protocol |
Mono-nucleosomal DNA from low MNase digestion is 1.5~2.5 % of bulk DNA. Mono-nucleosomal DNA from high MNase digestion is 25~30 % of bulk DNA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Animal use was approved by an IACUC committee. Mouse liver from C3H strain was minced with scalpels in SST (150 mM NaCl, 15 mM Na Citrate, 10 mM Tris [pH 7.4]) on ice, and washed with SST in 30 ml Corex tubes three times. The tissue was resuspended in RSB (10 mM Tris [pH7.4], 10 mM NaCl, 3 mM MgCl2, 10 mM sodium butyrate, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 1 mg/ml pepstatin) plus 0.5% NP40, and was swollen on ice for 5 min. The tissue was homogenized with a glass Teflon pestle in a glass homogenizer and a portion was observed under the microscope to confirm nuclear isolation. The nuclear suspension was filtered through a 100 μm cell strainer and pelleted by centrifugation at 1.4K rpm for 7 min at 4°C in 15 ml Corex tubes with a swinging bucket rotor. The pellet was washed in RSB plus 0.5% NP40 and pelleted by centrifugation at 900 rpm for 10 min at 4°C in 15 ml Corex tubes with a swinging bucket rotor, and this wash step was repeated one more time. The nuclear pellet was re-suspended in RSB and adjusted to O.D. A260 of 1. CaCl2 (3 mM final) was added, the nuclear suspension was pre-warmed for 1.5 min at 37°C, and was treated with MNase (Worthington Biochemicals) with 0 U/ml for control, 0.5 or 1 U/ml for low digestion and 20 U/ml for high digestion for 2 min at 37°C. Quench control (“Q” sample) was without MNase and CaCl2 and not warmed. The reactions were stopped by adding an equal volume of 2x TNESK (20 mM Tris [pH7.4], 200 mM NaCl, 2 mM EDTA, 2% SDS, 0.2 mg/ml Proteinase K), and incubated overnight at 37 °C. DNA was purified using phenol-chloroform extraction followed by ethanol precipitation. The nucleic acid was digested with 10 ng/ml of RNase for 20 min at 37°C and DNA was purified using phenol-chloroform extraction followed by ethanol precipitation. 50 μg samples of low MNase and 10 μg samples of high MNase were run on 6% polyacrylamide gel (TBE). The mono-nucleosomal DNA was excised from the 140 bp~200 bp size range. The gel was sliced into small pieces, put in 2 vol of diffusion buffer (500 mM ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS), and rotated overnight. The supernatant was filtered through Whatman GF/C and ethanol precipitated. DNA was purified using QIA EX II (QIAGEN) or AMPure XP (Beckman) beads. Mono-nucleosomal DNA from low MNase digestion averaged 1.5~2.5% of bulk DNA. Mono-nucleosomal DNA from high MNase digestion averaged 25~30% of bulk DNA. The percent of mononucleosomal DNA of bulk DNA was estimated based on the amount of mononucleosomal DNA recovered from a preparative PAGE gel, multiplied by 1.25 (PAGE gel extraction recovers about 80% of sample in our hands) compared to the amount of bulk DNA loaded onto to the PAGE gel.
We prepared multiplexing libraries from two biological replicates of 150 ng of low and high MNase digested, purified mono-nucleosomal DNA. We followed the standard Illumina multiplexing library preparation protocol with the following changes: 1) All QIAGEN purification steps were substituted with AMPure XP beads (Beckman) purifications. 2) For MNase digested mono-nucleosomal DNA, gel purification following adapter ligation was substituted with AMPure XP beads purification in order to remove excess adapters. 3) 10 ng of adapter ligated DNA was used as a template for PCR, wth 10 PCR cycles. 4) Gel purification after PCR was substituted with AMPure XP beads purification in order to remove excess primers. The DNA libraries were assessed using on an Agilent Technologies 2100 Bioanalyzer.
MNase digested DNA libraries were sequenced as 100-bp paired-end, using Illumina HiSeq2000.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
5094-0
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| Data processing |
Bases were called and quality control was performed using the Illumina pipeline (Casava)
Raw MNase-seq data were aligned with bowtie 0.12.5, using -m 1 and --best parameters.
Track views were obtained by reducing all MNase paired-end fragments (pooled across three technical replicates) to the central 140bp region and then binning into contiguous 10bp intervals.
MNase-seq calls were performed independently on low and high Mnase-seq datasets using GeneTrack (46) (sigma or s=40, exclusion zone or e =200). Using an exclusion zone of 200 bp prevents MNase-seq calls that are within 100 bp from each other. A 45 bp matching window was used to match MNase-seq calls across low and high MNase-seq datasets. Subsequently, EdgeR on 145bp window counts (centered around the MNase-seq call midpoints) was used to identify MNase hypersensitive regions that have significantly higher reads in low-MNase-seq datasets with respect to high-MNase-seq datasets.
Genome_build: mm8
Supplementary_files_format_and_content: BED files: *.merge.fragments.bed files show pooled replicate paired-end fragments (start of Watson tag to start of Crick strand), and the Low vs High file contains positions of hypersensitive nucleosomes; bigWig files: contains track views for each biological replicate (pooled across three technical replicates).
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| Submission date |
May 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
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| Department |
Cell & Developmental Biology
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| Lab |
Zaret Lab
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE57558 |
Hypersensitive Nucleosomes in Chromatin Are Intrinsic to the Structure of Active, Tissue-Specific Enhancers [MNase-Seq] |
| GSE57559 |
Hypersensitive Nucleosomes in Chromatin Are Intrinsic to the Structure of Active, Tissue-Specific Enhancers |
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| Relations |
| BioSample |
SAMN02769587 |
| SRA |
SRX540274 |
| 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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