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| Status |
Public on Mar 17, 2023 |
| Title |
O_350_rep1 |
| Sample type |
SRA |
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| Source name |
mouse liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: C57BL/6JN
salt concentration: 350
group: old
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| Treatment protocol |
Old as treated group
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei was released from frozen tissue by douncing in TM2 buffer (10 mM Tris HCl, pH 7.4, 2 mM MgCl2 supplemented with 1X Halt protease and phosphatase inhibitor and 0.5 mM PMSF), pelleted by centrifugation and washed once to remove debris. The washed pellet was resuspended in TM2 buffer with 1 mM CaCl2 and 12000U of MNase (New England Biolabs), incubated at 23°C for 15 minutes and the reactions stopped by addition of 0.5 mM EGTA. Approximately 30% was saved for analysis (“0”) and the rest was pelleted by centrifugation. The supernatant was removed and saved as “Supernatant” fraction after clearing. The nuclei were washed once with TM2 buffer and then resuspended in 70 µl Triton buffer (10 mM Tris–HCl pH 7.4, 2 mM MgCl2, 2 mM EGTA, 0.1% Triton X-100 supplemented with 1X Halt protease and phosphatase inhibitor and 0.5 mM PMSF) and evenly divided. The first aliquot was saved as “Input” while the second was used for sequential salt extraction by using Triton buffer with either 67.5, 150, 250 or 350 mM NaCl. Each extraction was done by incubating the resuspended pellet at 4°C for 2 hours. After each extraction, the nuclei were pelleted by centrifugation and the supernatant saved as “67.5”, “150”, “250” or “350” mM fraction. Finally, the remainder pellet which corresponds to ~5–10% of chromatin, was resuspended in 35 µl of TNE buffer (10 mM Tris–HCl pH 7.4, 200 mM NaCl, 1 mM EDTA supplemented with 1X Halt protease and phosphatase inhibitor) and labeled as “Pellet” fraction. DNA was purified from each of these fractions using DNA clean and concentrator columns (Zymo Research).
Purified DNA (~10 ng) was used to make libraries using the NEBNext Ultra II kit (New England Biolabs).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Library strategy: DNA-Seq
Illumina sequencing reads (~54 million paired end reads per sample) were de-multiplexed generating compressed FASTQ files by the on-board DRAGEN informatics pipeline (Illumina DRAGEN FASTQ Generation – 3.7.4) on the NextSeq 2000.
The FASTQ files were trimmed to remove adapter sequences with cutadapt/3.0 and the qualities of the FASTQs checked by running FASTQC/0.11.9. The reads were aligned with bowtie/2-2.4.2 using the end-to-end option to the GRCm38/mm10 genome assembly
Sam output files were then filtered to retain alignments with a minimum mapping quality of 2 using samtools/1.9. Reads mapping to Encyclopedia of DNA Elements (ENCODE) blacklist regions were removed from the analysis.
The bamCoverage option in deepTools/3.5.0 was used to generate RPKM (reads per kilobase per million mapped reads) normalized bigWig files. Input reads were subtracted from the different salt fractions and pellet with bigwigCompare.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated using bamCoverage in deepTools/3.5/0; Scores represent the coverage on the genome location
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| Submission date |
Oct 12, 2021 |
| Last update date |
Jun 28, 2023 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
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| Department |
Laboratory of Genetics and Genomics
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| Lab |
Computational Biology & Genomics Core
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| Street address |
251 Bayview Blvd
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE185707 |
A hyper-quiescent chromatin state is a barrier to productive regeneration during aging. [DNA-Seq] |
| GSE185708 |
A hyper-quiescent chromatin state is a barrier to productive regeneration during aging. |
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| Relations |
| BioSample |
SAMN22224164 |
| SRA |
SRX12575872 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5621873_O_350_rep1.bw |
168.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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