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| Status |
Public on Mar 17, 2023 |
| Title |
Y4_liver_EZH2_Input |
| Sample type |
SRA |
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| Source name |
mouse liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: C57BL/6JN
age group: young
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| Treatment protocol |
Old as treated group
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crude nuclei preparations were made from ~100 mg frozen tissue by douncing in nuclei preparation buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 0.1% NP-40, 0.01% digitonin and 1 mM BSA, supplemented with protease inhibitors and 1 mM sodium butyrate). After centrifugation, the nuclei pellet was crosslinked with 1% formaldehyde in 2 ml PBS by constant rotation at room temperature for 10 min followed by quenching with 125 mM glycine and two PBS washes. The nuclei were lysed with nuclei lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium-deoxycholate, 0.5% N-lauroylsarcosine supplemented with protease inhibitors and 1mM sodium butyrate) and sheared to <500bp using a Covaris S220 Ultrasonicator. An aliquot of the sheared chromatin sample was removed to check sonication success and to quantify the amount of chromatin. The immunoprecipitation, wash and elution steps were performed as reported previously (Sen et al., 2019) except ~4% HeLa chromatin was spiked in to 2 µg mouse chromatin prior to immunoprecipitation (only for liver samples).
DNA (~5 ng) was used to prepare libraries with the NEBNext Ultra II library preparation kit with unique dual index primers (New England Biolabs). The library quality and quantity were verified by BioAnalyzer DNA 1000 (Agilent) run and qPCR with NEBNext Library Quant kit (New England Biolabs) respectively.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Illumina sequencing reads (~50 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 trimgalore/0.6.6 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 separately to the GRCm38/mm10 and GRCh38/hg38 genome assemblies.
Sam output files were then filtered to keep alignments with a minimum mapping quality of 10 using samtools/1.9 and PCR duplicates removed with picard/2.23.7. Sambamba/0.7.1 was used to keep only uniquely aligned reads. Reads mapping to Encyclopedia of DNA Elements (ENCODE) blacklist regions were also removed from the analysis.
The bamCoverage option in deepTools/3.5.0 was used to generate RPKM normalized bigWig files. H3 and input reads were subtracted from H3K27me3 and IgG respectively with bigwigCompare.
Peaks were called using peakranger/1.18 with the bcp option using H3 and input as background for H3K27me3 and IgG respectively from the same animal
Assembly: mm10, hg38
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 |
Apr 14, 2022 |
| Last update date |
Mar 17, 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) |
| GSE185704 |
A hyper-quiescent chromatin state formed during aging is reversed by regeneration [ChIP-seq] |
| GSE185708 |
A hyper-quiescent chromatin state is a barrier to productive regeneration during aging. |
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| Relations |
| BioSample |
SAMN27585038 |
| SRA |
SRX14858066 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6044007_Y4_liver_EZH2_Input.bw |
163.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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