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Sample GSM2830115

Query DataSets for GSM2830115

Status

Public on Jan 12, 2018

Title

CF3P8

Sample type

SRA

Source name

Sperm

Organism

Rattus norvegicus

Characteristics

strain: Sprague-Dawley SR
cell type: sperm
treatment: Control
generation: F3
measurement: H3K27me3 methylation

Treatment protocol

On days 8 through 14 of gestation, the females were administered daily intraperitoneal injections of vinclozolin (100 mg/kg BW/day), or DDT (25 mg/kg BW/day) or dimethyl sulfoxide (DMSO) in oil (1 µl/kg BW/day vehicle). Vinclozolin and DDT (dichlorodiphenyltrichloroethane) were obtained from Chem Service Inc. (West Chester, PA). Vinclozolin and DDT were injected in DMSO vehicle as previously described. Treatment lineages are designated “control” or “vinclozolin” or “DDT” lineages. Five additional rats (samples HisS1, HisS2, HisS3, HisS5, HisS6) were neither treated nor bred. These rats provided samples that were analyzed individually, rather than being grouped together into pools.

Growth protocol

Female and male rats of an outbred Hsd: Sprague Dawley SR®TM (Harlan) at about 70 and 100 days of age were fed ad lib with a standard rat diet and ad lib tap water for drinking. To obtain time-pregnant females, the female rats in proestrus were pair-mated with male rats. The sperm-positive (day 0) rats were monitored for diestrus and body weight. The gestating female rats treated were designated as the F0 generation. The offspring of the F0 generation rats were the F1. Non-littermate females and males aged 70-90 days from F1 generation of control, vinclozolin or DDT were bred to obtain F2 generation offspring. The F2 generation rats were similarly bred to obtain the F3 generation offspring. Individuals were maintained for 120 days and euthanized for sperm collection. The F1-F3 generation offspring were not themselves treated directly with vinclozolin or DDT. The control, vinclozolin and DDT lineage rats were housed in the same room with lighting, food and water as previously described

Extracted molecule

genomic DNA

Extraction protocol

The epididymis was dissected free of connective tissue, a small cut made to the cauda and tissue placed in 3 ml of phosphate buffered saline (PBS) for 20 minutes at room temperature and then kept at 4ºC to immobilize the sperm. The epididymal tissue was minced and then the released sperm centrifuged at 11,000 x g and the pellet resuspended in NIM buffer and stored at -80ºC until processed further. To isolate DNA, 100 µl of sperm suspension was combined with 820 µl DNA extraction buffer 1M Tris HC1, pH 8.0; 0.5M EDTA (ethylenediaminetetraacetic acid); 10% SDS (sodium dodecyl sulfate) and 80 µl 0.1M DTT (dithiothreitol). The sample was incubated at 65ºC for 15 minutes. Following this incubation, 80 µl proteinase K (20 mg/ml) was added and the sample incubated at 55ºC for at least 2 hours under constant rotation. Then 300 µl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 13,500 rpm for 20 min at 4ºC. 1 ml of the supernatant was transferred to a 2 ml tube and 2 µl of Glycoblue (Invitrogen, Carlsbad, CA) and 1 ml of cold 100% isopropanol were added. The sample was mixed well by inverting the tube several times then left in -20ºC freezer for at least one hour. After precipitation, the sample was centrifuged at 13,500 rpm for 20 min at 4ºC. The supernatant was taken off and discarded without disturbing the blue pellet. The pellet was washed with 70% cold ethanol by adding 500 µl of 70% ethanol to the pellet and returning the tube to the freezer for 20 min. After the incubation, the tube was centrifuged for 10 min at 4ºC at 13,500 rpm and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and then a gel loading tip was used to remove as much liquid as possible. Pellet was air-dried at RT until it looked dry (about 5 min). Pellet was then resuspended in 100 µl of nuclease free water. Equal amounts of DNA from individual sperm samples were used to produce three DNA pools for histone chromatin immunoprecipitation (ChIP). The antibody used for the H3 location ChIP was Anti-Histone H3, Ct, pan, clone A3S, manufactured by Millipore (Cat. #05-928, Lot #2780491). The antibody used for the H3K27me3 ChIP was Anti-Trymethyl-Histone H3 (Lys27 polyclonal antibody) manufactured by Millipore (Cat. #07-449, Lot #2826067).
The ChIP pools were used to create libraries for next generation sequencing (NGS) using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB, San Diego, CA). The manufacturer protocol was followed.
Each pool received a separate index primer. NGS was performed at the WSU Spokane Genomics Core using Ilumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 35 million reads per pool. Six libraries were run in one lane.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Sample name: sP8
processed data file:F3me.DDT.results.csv.gz

Data processing

Basic read quality was verified using summaries produced by FastQC
The reads for each sample were mapped to the rat (Rattus norvegicus) genome using Bowtie2
The mapped read files were then converted to sorted BAM files using SAMtools
The MEDIPS R package was used to calculate differential coverage between sample groups. To identify DHR, the reference genome was broken into 100 bp windows. The edgeR Pvalue was used to determine the relative difference between the two sample groups for each genomic window. Windows with an edgeR P value less than 1e-7 (DDT) or 1e-6 (vinclozolin) were considered DHR. The DHR edges were extended until no genomic window with an edgeR P value less than 0.1 remained within 1000 bp of the DHR. In addition to the DHR analysis, the core histones were identified using a raw read depth threshold of 150 reads. Core site edges were extended until no genomic window with a read depth > 150 remained within 1000 bp of the site.
The DHR that included at least one genomic window with an edgeR P value < 1e-6 (DDT) or 1e-7 (vinclozolin) were then selected for further analysis and annotated.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: The results of the edgeR analysis in CSV format. This includes raw read counts for each genomic window as well as the calculated p-value and other summary values. The five.rats.results.csv.gz file includes only raw read depth counts and RPKM adjusted counts for each genomic window since no p-values were calculated.

Submission date

Oct 24, 2017

Last update date

May 15, 2019

Contact name

Michael K Skinner

E-mail(s)

[email protected]

Organization name

WSU

Department

SBS