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| Status |
Public on Mar 17, 2023 |
| Title |
O4_liver_5mC |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: C57BL/6JN
age: 82 weeks
sex (m=male, f=female): M
medip antibody: 5mC (Diagenode, 33D3 clone, C15200081, Lot RD-004)
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| Treatment protocol |
Old as treated group
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
meDIP-seq was performed using the MagMeDIP-seq Package (Diagenode) following the manufacturer’s protocol with a mouse antibody against 5mC (Diagenode, 33D3 clone, C15200081). Briefly, 1.2 µg of genomic DNA was sonicated into ~200 bp fragments using the S220 focused ultrasonicator (Covaris). Prior to immunoprecipitation, samples were spiked with hydroxymethylated, methylated and unmethylated internal DNA controls. IP efficiency and success was verified by qPCR targeting internal DNA controls. The DNA amount was quantified by Qubit HS DNA kit and the fragment size was assessed on a 2100 BioAnalyzer using a DNA HS kit (Agilent).
Individual libraries for immunoprecipitated DNA and 10% input were dual indexed (NEBNext Multiplex Oligos, unique dual indices, New England Biolabs), PCR amplified and then pooled into equimolar amounts and further quantified using the NEBNext Library Quant Kit (New England Biolabs). The pooled library was subjected to 50 bp paired-end sequencing on the Illumina NextSeq 2000 platform.
MagMeDIP-seq
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| 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 genome assembly.
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. Input reads were subtracted from MeDIP samples 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
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 |
Jan 23, 2023 |
| 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) |
| GSE185708 |
A hyper-quiescent chromatin state is a barrier to productive regeneration during aging. |
| GSE223480 |
A hyper-quiescent chromatin state formed during aging is reversed by regeneration |
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| Relations |
| BioSample |
SAMN32875472 |
| SRA |
SRX19141933 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6957402_O4_liver_5mC.bw |
82.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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