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| Status |
Public on Feb 24, 2017 |
| Title |
DR 26m PE Rep1 BS-seq |
| Sample type |
SRA |
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| Source name |
DR 26m PE BS-seq
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| Organism |
Mus musculus |
| Characteristics |
strain: C3B6F1
gender: female
diet: dietary-restricted (DR)
age: 26 months
tissue: liver
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| Treatment protocol |
We used females of the long-lived F1 hybrid mouse strain (C3B6F1), which responds to DR with a robust increase in lifespan. To avoid effects on post-natal development, DR treatment was started in 12-week-old animals, by providing DR mice with 40% less food than their AL controls. We collected liver samples of AL and DR animals at the ages of 5 months (young) and 26 months (old).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA-seq: RNA from 3 AL and 3 DR animals per time point was isolated from 30 mg of liver tissue using Trizol Reagent (ThermoFisher) according to the manufacturer’s instructions, followed by DNAse treatment with the TURBO DNA-free Kit (ThermoFisher). RNA integrity was analyzed using the Agilent TapeStation System.
BS-seq: DNA of 3 AL and 3 DR animals per timepoint was isolated from 30 mg of liver tissue using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions.
RNA-seq: TruSeq RNA-seq library preparation was conducted as previously described (Sultan, et al., 2012) using 3 µg of total RNA as input and rRNA depletion. Barcoded libraries were sequenced with 2x40 mio, 100 bp paired-end reads on an Illumina HiSeq2500.
BS-seq: Around 400 ng genomic DNA was used as input for BS-Seq library generation. DNA was sonicated, and end repair and A-tailing were performed using the NEB Next kit according to the manufacturers’ instructions. Illumina’s Early Access Methylation Adaptor Oligo Kit was used for adaptor ligation. Adaptor-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturer’s instructions for the one-step protocol. Bisulfite-treated DNA was amplified using PfuTurbo Cx Hotstart DNA Polymerase from Agilent Technologies with 14–18 cycles depending on input amount. Size selection was performed by gel extraction for DNA fragments between 200 bp and 250 bp, as adapted from (Seisenberger et al., 2012). Libraries of young samples were sequenced using the paired-end protocol. Old sample libraries were sequenced using single-end protocol and a subsequent re-run using a paired-end protocol to increase coverage.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Dietary restricted
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| Data processing |
RNA-seq: Raw sequence reads were trimmed to remove adaptor contamination and poor-quality reads using Trim Galore! (v0.3.7, parameters: --paired --length 25). A modified mouse reference genome (build GRCm38), with all known SNPs replaced by ‘N’ was set up to account for the hybrid genome of the C3H-C56BL/6 mice. Trimmed sequences were aligned using Tophat2 (v2.0.14, parameters: --no-mixed --library-type=fr-firststrand -g 2 -p 15 -r 500 --mate-std-dev 525), supplying GENCODE annotation (release M9, main annotation) for improved mapping. Multi-mapped reads were filtered using samtools (v1.2, parameters: view -F 0x100 -b –h). Data visualization and analysis was performed using SeqMonk, ClueGO {Bindea:2009hh} custom RStudio and the following Bioconductor packages: Deseq2, topGO and org.Mm.eg.db.
BS-seq: Raw sequence reads were trimmed using Trim Galore (v0.4.2; default parameters). Trimmed sequences were aligned using Bismark (v0.16.3; default parameters). Methylation calls were extracted after duplicate sequences had been excluded (parameters --ignore_r2 2 for paired-end libraries).
Genome_build: GRCm38
Supplementary_files_format_and_content: RNA-seq: tab-delimited text file include raw counts for each sample; results for differential gene expression analysis.
Supplementary_files_format_and_content: BS-seq: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
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| Submission date |
Dec 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Oliver Hahn |
| E-mail(s) |
ohahn@calicolabs.com
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| Organization name |
Calico Life Sciences, LLC
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| Lab |
Oliver Hahn
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| Street address |
1170 Veterans Blvd
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE92486 |
Dietary restriction protects from age-associated DNA methylation and induces epigenetic reprogramming of lipid metabolism |
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| Relations |
| BioSample |
SAMN06158680 |
| SRA |
SRX2431060 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2430573_DR_5m_PE_Rep1_BS-seq.cov.txt.gz |
168.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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