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| Status |
Public on Jun 05, 2018 |
| Title |
P2_antiH3_ChIP |
| Sample type |
SRA |
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| Source name |
Sperm
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley SR
cell type: Sperm
treatment: Control
generation: F1
measurement: ChIP-seq
ip antibody: monoclonal rabbit antihistone H3, Millipore 05-928
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| Treatment protocol |
On days 8 through 14 of gestation, the females were administered daily intraperitoneal injections of vinclozolin (100 mg/kg BW/day) or dimethyl sulfoxide (DMSO). DDT (dichlorodiphenyltrichloroethane) was obtained from Chem Service Inc. (West Chester, PA). Vinclozolin was obtained from Chem Service Inc. (West Chester, PA) and was injected in a 20 microliter DMSO vehicle.
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| 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 or vinclozolin 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. The control and vinclozolin lineage rats were housed in the same room with lighting, food and water.
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| 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 5 ml of 1x PBS solution for up to 24 hours at 4°C. The epididymal tissue was minced and the released sperm was centrifuged at 6,000 x g, then the supernant removed, and the pellet resuspended in NIM buffer, to be stored at -80O C until further use. One hundred µl of sperm suspension was sonicated, spun down at 6,000 g, the pellet washed with 1x PBS once, and then combined with 820 µL DNA extraction buffer and 80 µl 0.1M DTT. 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, A7953) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 14,000 rpm for 30 minutes at 4ºC. One ml of the supernatant was transferred to a 2 ml tube and 2 µl of glycoblue 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 14,000 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 minutes. After the incubation the tube was centrifuged for 10 min at 4ºC at 14,000 rpm and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and then as much liquid as possible was removed with gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 minutes). Pellet was then resuspended in 100 µl of nuclease free water. Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: rat sperm DNA pools were generated using the appropriate amount of genomic DNA from each individual for 3 pools each of control and vinclozolin lineage animals. Genomic DNA was sonicated using the Covaris M220 the following way: the pooled genomic DNA was diluted to 130 μl with TE buffer into the appropriate Covaris tube. Covaris was set to 300 bp program and the program was run for each tube in the experiment. 10 μl of each sonicated DNA was run on 1.5% agarose gel to verify fragment size. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube and the volume measured. The sonicated DNA was then diluted with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) to 400 μl, heat-denatured for 10 min at 95˚C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4˚C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 mL 1XPBS with 0.1% BSA and 2mM EDTA) was added and the bead sample was resuspended. Tube was then placed into a magnetic rack for 1-2 minutes and the supernatant discarded. The tube was removed from the magnetic rack and the beads washed once. The washed beads were resuspended in the same volume of 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl of DNA-antibody mixture from the overnight incubation, then incubated for 2h on a rotator at 4˚C. After the incubation the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into magnetic rack for 1-2 minutes and the supernatant discarded, then washed with 1xIP buffer 3 times. The washed bead-DNA solution is then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/ml). The sample was then incubated for 2-3 hours on a rotator at 55˚ C and then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the supe and the tube vortexed for 30 sec then centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20˚C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20min at 4˚C and the supernatant removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20˚C freezer for 15 min. then centrifuged again at 14,000rpm for 5min at 4˚C and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and as much liquid as possible was removed with gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 minutes) then resuspended in 20μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212).Histone chromatin immunoprecipitation with genomic DNA was performed as follows: rat sperm pools were generated using a total of 8 million sperm per pool for 3 pools of control and vinclozolin lineage animals. The control pools contained equal amounts of sperm for each of 3-4 individuals for a total of n=11 rats and the vinclozolin pools contained equal amounts of sperm for each of 3 individuals for a total of n=9 rats per exposure group. To remove any somatic cell contamination sperm samples from each animal were sonicated 10 seconds using a Sonic Dismembrator Model 300 (Thermo Scientific Fisher, USA) then centrifuged 4,000xg for 5 min at 4°C. The supernatant was discarded and the pellet resuspended and counted individually on a Neubauer counting chamber (Propper Manufacturing Co., Inc., New York, USA) prior to pooling. The sperm pools were reconstituted up to 1 ml with 1X PBS. To reduce disulfide bonds, 50 μl of 1M DTT was added to each pool and the pools were then incubated for 2 hours at room temperature under constant rotation. To quench any residual DTT in the reaction, 120 μl of fresh 1M NEM (N-Ethylmaleimide, Thermo Scientific, Rockford, USA) was then added and the samples incubated for 30 min at room temperature under constant rotation. The sperm cells were pelleted at 2,000g for 5 min at room temperature and the supernatant discarded. Pellets were resuspended in 1X PBS and then spun again at 2,000g for 5 min at room temperature. The supernatant was discarded. The sperm cells were then resuspended in “buffer 1” (final concentration: 15 mM Tris-HCl (pH 7.5), 60 mM KCl, 5 mM MgCl2, 0.1 mM EGTA; all filtered through a 0.22 μm filter and stored at room temperature) in a ratio of 2 million sperm cells per 50 μl. “Complete buffer” was “buffer 1” supplemented with 0.5% tergitol (vol/vol) and 1% DOC (wt/vol) (sodium deoxycholate, Sigma Aldrich 30970). 50 μl of this supplemented buffer was added to each aliquot. The samples were mixed and incubated for 20 min on ice. Sperm cell DNA was divided into aliquots of 4 μg of DNA. These aliquots were sonicated using the Covaris M220 the following way: 4 μg of genomic DNA was resuspended in 130 μl of complete buffer supplemented with tergitol 0.5% and DOC 1%. Covaris was set to a 10 min “Chromatin shearing” program and the program was run for each tube in the experiment. For each sample 10 μl was run on a 1.5% agarose gel to verify fragment size.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
processed data file:
F1.DHR.vin.csv.gz
The complete extraction protocol is available in a pdf on the series record.
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| Data processing |
Basic read quality was verified using summaries produced by FastQC. The raw reads were trimmed and filtered using Trimmomatic. The reads for each MeDIP and ChIP sample were mapped to the Rnor 6.0 rat genome using Bowtie2 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools.
To identify DMRs and DHRs, the reference genome was broken into 100 bp windows. Genomic windows with less than 40 mapped reads summed across all samples were removed prior to further analysis. The MEDIPS R package was then used to calculate differential coverage between control and exposure sample groups. The edgeR p-value was used to determine the relative difference between the two groups for each genomic window.
Windows with an edgeR p-value less than 10-6 were considered DMRs or DHRs. The DMR/DHR edges were extended until no genomic window with a p-value less than 0.1 remained within 1000 bp of the DMR/DHR.
CpG density and other information was then calculated for the DMR/DHR based on the reference genome.
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.
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| Submission date |
Mar 05, 2018 |
| Last update date |
Jun 05, 2018 |
| Contact name |
Michael K Skinner |
| E-mail(s) |
skinner@mail.wsu.edu
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| Organization name |
WSU
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| Department |
SBS
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| Street address |
Abelson 507
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| City |
Pullman |
| State/province |
WA |
| ZIP/Postal code |
99163 |
| Country |
USA |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE111441 |
Alterations in sperm DNA Methylation, Non-Coding RNA expression, and histone retention mediate Vinclozolin induced epigenetic transgenerational inheritance of disease |
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| Relations |
| BioSample |
SAMN08637656 |
| SRA |
SRX3766354 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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