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Sample GSM2816992

Query DataSets for GSM2816992

Status

Public on Nov 10, 2017

Title

ATACseq TBT rep 7

Sample type

SRA

Source name

Sperm

Organism

Mus musculus

Characteristics

strain: C57BL/6J
age: 8 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

Mouse sperm were isolated from 6 randomly selected, non-sibling males per generation. Epididymis and vas deferens were dissected and placed on a petri dish containing 2 mL of nucleus isolation medium (NIM) buffer (123.0 mmol/l KCl, 2.6 mmol/l NaCl, 7.8 mmol/l NaH2PO4, 1.4 mmol/l KH2PO4 and 3 mmol/l EDTA (disodium salt)). Sperm was pushed out of the tissue and media was collected and transferred to two clean microcentrifuge tubes and kept on ice. Samples were centrifuged at 13,000 xg for 5 minutes at 4ºC. Supernatant was removed and cells were resuspended in 1 mL of fresh NIM buffer.
Sperm were counted using a hemocytometer, and 50,000 sperm were suspended in 22.5 uL water containing 0.01% digitonin. The diluted sperm were immediately subjected to tagmentation by adding 25 uL Nextera tagment DNA buffer (2X reaction buffer from Illumina Nextera kit) and 2.5 uL Nextera tagment DNA enzyme (Tn5 transposon enzyme from Nextera kit) followed by incubation at 37 °C for 30 minutes. Tagmented sperm DNA was immediately purified using QIAGEN MinElute kit and subjected to PCR amplification of the tagmented DNA fragments for Illumina deep sequencing library construction as previously described. To avoid batch effects, we randomly selected one sperm specimen from each of the four treatment groups – namely, F3 DMSO, F3 TBT, F4 DMSO, and F4 TBT – and performed the ATAC-seq reaction for these four specimens as a batch on the same day. We processed six batches (total 24 sperm specimens, 6 specimens for each treatment group) as independently performed experiments. The ATAC-seq libraries were sequenced using Illumina NextSeq 500 sequencer

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

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