GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2816968

Query DataSets for GSM2816968

Status

Public on Nov 10, 2017

Title

MBDseq DMSO rep 3

Sample type

SRA

Source name

Gonadal adipose tissue

Organism

Mus musculus

Characteristics

strain: C57BL/6J
age: 33 weeks
generation: F4

Treatment protocol

Female C57BL/6J mice (25 females per treatment group) were randomly assigned to the different treatment groups and exposed via drinking water to 50 nM TBT or 0.1% DMSO vehicle (both diluted in 0.5% carboxymethyl cellulose in water to maximize solubility) for 7 days prior to mating. Chemicals were administered to the dams throughout pregnancy and lactation.We chose only 1 male and 2 females per litter for breeding. To randomize the breeding process as much as possible, we did not breed siblings and did not breed females from the same litter with the same male. Unexposed animals were bred to each other and TBT-exposed bred to each other. A similar approach was followed in generations F2-F4. Animals from F0-F4 were maintained on a standard diet (SD - Rodent Diet 20 5053*; PicoLab) throughout the experiment. F4 males and females (avoiding siblings within the same gender) were maintained on a higher fat diet (HFD - Mouse Diet 20 5058*; PicoLab) between weeks 19-25. Mice were subsequently returned to the SD for 8 more weeks prior to euthanasia at 33 weeks of age

Growth protocol

Male (n=25) and female (n=50) C57BL/6J mice (7 weeks of age) were purchased from the Jackson Laboratory (Sacramento, CA). Mice were housed in micro-isolator cages in a temperature-controlled room (21–22°C) with a 12 hr light/dark cycle. Water and food was provided ad libitum unless otherwise indicated. Animals were treated humanely and with regard for alleviation of suffering. All procedures conducted in this study were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. All tissue harvesting was performed with the dissector blinded to which groups the animals belonged to.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA (gDNA) and RNA were isolated from male white gonadal adipose tissue using ZR-Duet™ DNA/RNA MiniPrep kit (Zymo Research) of animals sacrificed at 33 weeks of age (after the diet challenge). Nucleic acid samples from 4 randomly selected mice from control or TBT groups were submitted for DNA methylome and transcriptome analyses
Sonicated gDNA (100-200 bp, 500 ng) was subjected to methylated DNA enrichment using the MethylMiner kit (Life Technologies), followed by deep sequencing library construction using the NEBNext Ultra kit (New England Biolabs). Sonicated gDNA without methylation enrichment was used as input control. Following the ENCODE recommendation for ChIP-seq, we aimed for 20 million mapped reads (75 nt single) for each gDNA sample

Library strategy

MBD-Seq

Library source

genomic

Library selection

MBD2 protein methyl-CpG binding domain

Instrument model

Illumina NextSeq 500

Description

MBD-seq_processed data.txt

Data processing

Statistical evaluation of TBT-dependent methylome and transcriptome variation in F4 male gonadal white adipose tissue was performed using R (version 3.3), and Bioconductor (version 3.3) packages MEDIPS (version 1.22), Rsubread (version 1.22), and edgeR (version 3.14). MEDIPS function MEDIPS.meth was used to estimate the statistical significance of MBD-seq reads differential coverage for GRCm38/mm10 mouse reference genomic 100-bp consecutive, non-overlapping windows with the following parameters: diff.method = edgeR, minRowSum = 10, and diffnorm = quantile. Rsubread function featureCounts was used to assigned uniquely mapped RNA-seq reads to GRCm38/mm10 mouse reference genome gene models and count reads with the following parameters: GTF.featureType = exon, GTF.attrType = gene_id, allowMultiOverlap = TRUE, nthreads = 24, and strandSpecific = 0 . edgeR functions cpm and glmQLFTest were used to estimate the number of counts per million per gene model and the statistical significance of RNA-seq reads differential coverage.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: RNA-seq_processed data.txt includes RNA-seq normalized counts per gene model for each sample, and results of the statistical analyses performed as described above.
Supplementary_files_format_and_content: MBD-seq_processed data.txt includes MBD-seq read counts, and rpkm for each sample, and results of the statistical analyses performed as described above for GRCm38/mm10 mouse reference genomic 100-bp consecutive, non-overlapping windows for which P value was lower than 0.001.
Supplementary_files_format_and_content: ATAC-seq_isochores_f3_processed data.txt and ATAC-seq_isochores_f4_processed data.txt include ATAC-seq read counts, and rpkm for each sample, and results of the statistical analyses performed as described above for GRCm38/mm10 mouse isochores obtained from isoFinder for sperm samples from F3 and F4 generations, respectively. ATAC-seq_SICER_f3_processed data.txt and ATAC-seq_SICER_f4_processed data.txt include ATAC-seq read counts, and normalized counts for each group of samples, and results of the statistical analyses performed as described above for GRCm38/mm10 mouse SICER islands for sperm samples from F3 and F4 generations, respectively.

Submission date

Oct 16, 2017

Last update date

May 15, 2019

Contact name

Raquel Chamorro-Garcia

E-mail(s)

rchamorr@ucsc.edu

Organization name

University of California Irvine

Street address

4351 Natural Sciences 2