|
| Status |
Public on Aug 08, 2017 |
| Title |
M40 |
| Sample type |
SRA |
| |
|
| Source name |
atrazine_F3_Sperm
|
| Organism |
Rattus norvegicus |
| Characteristics |
treatment: atrazine
generation: F3
pools or individuals: individual
tissue: Sperm
|
| Treatment protocol |
On days 8 through 14 of gestation, the F0 females were administered daily intraperitoneal injections of Atrazine (25 mg/kg BW/day) or dimethyl sulfoxide (vehicle). The atrazine was obtained from Chem Service, Westchester PA and was injected in approximately 200 microliters of DMSO vehicle as previously described. The F1- F3 generation offspring were not themselves treated directly with atrazine.
|
| Growth protocol |
Female and male rats of an outbred strain Hsd:Sprague Dawley®™SD®™ (Harlan) at about 70 to 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 changes in body weight. The gestating female rats treated were designated as the F0 generation. The offspring of the F0 generation rats were the F1 generation. Non-littermate females and males aged 70-90 days from F1 generation control or atrazine lineages were bred to obtain F2 generation offspring. The F2 generation rats were bred to obtain F3 generation offspring.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fifty to hundred ul of rat sperm suspension were used for DNA extraction, then 820 uL DNA extraction buffer and 80 ul 0.1M DTT added. The sample was incubated at 65C for 15 minutes. Following this incubation 80 ul proteinase K (20 mg/ml) was added and the sample was incubated at 55C for 2 hours under constant rotation. Then 300 ul of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) were added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 13,500 rpm for 20 minutes at 4C. One ml of the supernatant was transferred to a 2 ml tube and 2 ul 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 -20C freezer for at least one hour. After precipitation the sample was centrifuged at 13,500 rpm for 20 min at 4C. The supernatant was taken off and discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol by adding 500ul 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 4C at 13,500 rpm and the supernatant discarded. The tube was spun again briefly to collect residual ethanol at 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 ul of nuclease free water. For F1 and F2 generations equal amounts of DNA from each individual’s sperm sample was used to produce three different DNA pools per lineage and the pooled DNA used for methylated DNA immunoprecipitation (MeDIP). For F3 generation each individual’s sperm sample was analyzed separately for MeDIP-Seq.
The MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (San Diego, CA) starting at step 1.4 of the manufacturer’s protocol to generate double stranded DNA. After this step the manufacturer’s protocol was followed.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
processed data file:
F3.results.csv
|
| Data processing |
Basic read quality was verified using summaries produced by FastQC. Trimmomatic was used to remove low quality reads and bases
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 DMR, 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 an arbitrarily chosen threshold were considered DMR. The DMR edges were extended until no genomic window with an edgeR P value less than 0.1 remained within 1000 bp of the DMR.
The selected DMR were then summarized 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.
|
| |
|
| Submission date |
May 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael K Skinner |
| E-mail(s) |
skinner@mail.wsu.edu
|
| Organization name |
WSU
|
| Department |
SBS
|
| Street address |
Abelson 507
|
| City |
Pullman |
| State/province |
WA |
| ZIP/Postal code |
99163 |
| Country |
USA |
| |
|
| Platform ID |
GPL18694 |
| Series (1) |
| GSE98683 |
Atrazine Induced Epigenetic Transgenerational Inheritance of Disease, Lean Phenotype and Sperm Epimutation Pathology Biomarkers |
|
| Relations |
| BioSample |
SAMN06919991 |
| SRA |
SRX2792215 |
