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| Status |
Public on Apr 26, 2024 |
| Title |
rat testis, MEDIP nicotine 2 |
| Sample type |
SRA |
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| Source name |
testis
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| Organism |
Rattus norvegicus |
| Characteristics |
tissue: testis
age: adult 11-week-old
Sex: male
strain: Sprague-Dawley
treatment: MBD2-Fc
treatment: nicotine
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| Treatment protocol |
Using a sterile technique, a 28-day osmotic mini-pump (2ML4-ALZET, Durect Corp., Cupertino, CA, USA) filled with either sterile saline (control; CON) or a nicotine tartrate (6 mg/kg; NIC), dissolved in sterile saline was placed subcutaneously between the scapulae, as described before (Boychuk and Hayward 2011)(Chang, Karatayev, and Leibowitz 2013). On GD19, or 13 days following pump surgery, dams were allowed to give birth.
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| Growth protocol |
Specific pathogen-free male and female Sprague Dawley rats were purchased from Envigo (USA). Females were purchased at 8 weeks of age and were allowed 1 week to acclimate. At 9 weeks of age, females were randomly assigned to one of 4 treatment groups. Two groups of females were mated, and two groups of females remained virgins. Mating included placing a single female overnight with a male and examination on the following morning for the presence of sperm via vaginal swabs. If sperm was present, the dam was weighed and placed in a separate cage, and this period was marked as gestational day (GD) 0.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from 6 biological replicates using DNA Easy Blood and Tissue Kit (Qiagen, 69506) according to instructions provided by manufacturer. DNA concentration was measured by fluorescent method using fluorescent reader (Promega). 10 µgs of DNA was used for MeDIP using methylated DNA Enrichment Kit (NEB, #E2600S). DNA was diluted and sonicated in Qsonica sonicator using following conditions: 20 s pulse on, 20 sec pulses off, total sonication time is 8 min. After that, sonicated DNA was incubated with MBD2-Fc -protein A Magnetic beads. The beads were washed 4 times with 1XBind/Wash Buffer. The enriched methylated DNA was eluted from beads with miliQ water and and the concentration of methylated DNA was again determined by fluorescence.
6 ng of methylated DNA or input were taken for library preparation. Sequencing libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (E7645S; NEB). We performed 15 cycles for the library amplification.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Libraries were sequenced on Illumina NextSeq 2000 sequencer in a paired-end read mode of 50 base reads. Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq None.
The reads were mapped to the reference genome rn7 using Bowtie2 v2.5.0. The numbers of mapped reads were normalized by a scale factor to adjust the total number of reads. From the aligned reads, DNA methylated peaks were identified using 6 biological replicates and the corresponding input by MACS2 (2.2.7.1) algorithm; the following parameters were applied: a shift-size window of 73 bp, no model, and q-value threshold <0.05.
To compare the control datasets of the thia-exposed and control samples, differential peaks were identified using counting reads at each peak using bedtools MultiCovBed (Version 2.30.0). Statistical significance was calculated using Limma v3.50.1 with filtering peaks with low counts.
Each normalised bam file was converted to BedGraph tracks by using the Genome Coverage 2.30.0. All analyses were performed using Galaxy platform, Nucleic Acids Research, Volume 50, Issue W1, 5 July 2022, Pages W345–W351.
Assembly: rn7
Supplementary files format and content: bedgraph
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| Submission date |
Jan 18, 2024 |
| Last update date |
Apr 26, 2024 |
| Contact name |
Fatima SMAGULOVA |
| E-mail(s) |
fatima.smagulova@inserm.fr
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| Phone |
+3323233662
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| Organization name |
Irset - U1085
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| Lab |
Chromatin, Epigenetics, Environment
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| Street address |
9 av. Prof. Léon Bernard
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| City |
Rennes |
| ZIP/Postal code |
35000 |
| Country |
France |
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| Platform ID |
GPL32190 |
| Series (2) |
| GSE253594 |
The prenatal nicotine exposure leads to epigenetic alterations in nervous system signaling genes in the rat [MeDIP] |
| GSE253596 |
The prenatal nicotine exposure leads to epigenetic alterations in nervous system signaling genes in the rat |
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| Relations |
| BioSample |
SAMN39486233 |
| SRA |
SRX23276806 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8023948_FMSV487.bedgraph.gz |
115.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
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