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Sample GSM4733001

Query DataSets for GSM4733001

Status

Public on Nov 19, 2020

Title

M15_Sperm_Control_ChIP

Sample type

SRA

Source name

Sperm

Organism

Rattus norvegicus

Characteristics

strain: Sprague Dawley
Sex: Male
tissue: Sperm
treatment: Atrazine
measurement: ChIP
disease: obese
lineage: Control

Treatment protocol

Outbred Sprague Dawley gestating female rats (F0 generation) were administered an intraperitoneal dose of 25 mg/kg body weight of atrazine (4% of rat oral LD50 [41] and 50% of NOAEL). These doses were administered at 90 days of age, during embryonic days 8-14 (E8-E14) of fetal gonadal sex determination. The F1 generation offspring was directly exposed as a fetus and F2 generation grand-offspring exposed as the germline in the F1 generation. These were each bred at 90 days of age within the lineage. The F3 generation great-grand-offspring is required to establish the transgenerational inheritance generation of ancestral exposure. A control lineage was established that used F0 gestating rats exposed to the vehicle control dimethyl sulfoxide (DMSO).

Growth protocol

Female and male rats of an outbred strain Hsd:Sprague Dawley SD (Harlan) at 70 to 100 days of age were fed ad lib with a standard rat diet and ad lib tap water. The gestating female rats treated were designated as the F0 generation. F1- F3 generation control and atrazine lineages were housed in the same room and racks with lighting, food and water. All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee (protocol IACUC # 6252).

Extracted molecule

genomic DNA

Extraction protocol

For each sample, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4ºC will immobilize the sperm. The tissue was then minced, the released sperm pelleted at 4ºC 3,000 x g for 10 min, then resuspended in NIM buffer and stored at -80ºC for further processing. An appropriate amount of rat sperm suspension was used for ChIP analysis. Rat sperm samples were sonicated and then pretreated with 50 µl 1M DTT in each sample tube. Samples were then incubated for 2h at RT on rotation. 123 mg of NEM dissolved in 1 mL of milliQ water in a 56ºC incubator. 120 µL of NEM added to each sample and then incubated for 30 min at RT on rotation. Chromatin prepared with 400 µl "Buffer 1 Stock" (see cited ChIP protocol), 102 mg sucrose and 0.5 µl 1M DTT. Samples then centrifuged at 2,500 rpm 5 min at RT. Supernatant discarded. Sperm resuspended in 1 ml 1X PBS and centrifuged at 2,500 rpm 5 min at RT. Supernatant discarded. Sperm resuspended in 130 µl of “Complete Buffer” (“Buffer 1 stock” + detergents) and placed in 130 µl Covaris tubes. Incubate 20 minutes on ice. Covaris program set to to the desired program: Chromatin shearing 10 min, Peak power: 75.0, Duty factor: 10.0, Cycles/Burst: 200. Program run for each tube. Samples removed from Covaris tube and placed it in 1.5 ml tube and centrifuged at 12,500 rpm 10 min at RT. Supernatant then collected and protease inhibitor cocktail ((20X) by dissolving 1 tablet in 500 µl MilliQ H2O) is added (11 µl of this solution per sample). Samples run on aa 1.5 % agarose gel to check bands size before resuming the IP protocol. Each sample had 3 µl of antibody (Ab anti-histone H3, CT, pan, clone A3S Millipore cat #05-928) added and were placed at 4ºC on rotation O/N. Samples then washed with magnetic beads (ChIP grade protein magnetic beads, Cell Signal, at 4ºC). 40 µl of beads for each sample were washed: placed on DynaMag-2 for two minutes and supernatant removed without disturbing the beads. Resuspended with 1 ml of 1X IP buffer, then repeat wash process once. Supernatant removed and resuspended in 350 µl of 1x IP. 40 µl of beads added to each sample and placed on rotation at 4ºC for 3 h. Tubes were then washed on DynaMag with 1X IP 3-4 times. With the final wash, beads resuspended in 300 µl “MeDIP digestion buffer." 3 µl of Proteinase K 20 mg/ml added to each tube and placed on rotation at 56ºC, 4 h (or O/N). Samples centrifuged quickly, then supernatant placed in new 1.5 ml tubes. 300 µl of phenol/chloroform/isoamyalcohol added to each tube and Centrifuged at 12,500 rpm 10 min at RT. The aqueous phase was placed in a new 1.5 ml tube and 2 µl of Glycoblue, one tenth vol of 3 M sodium acetate (NaOAc) and 2 vol of ethanol were added to each sample. Samples then precipitated O/N at 20ºC (O/N precipitation necessary for better yields). Tubes centrifuged at 12,500 rpm 30 min at 4ºC. Supernatant removed and 500 µl of 70% EtOH added. Centrifuged at 12,500 rpm 10 min at 4ºC. Supernatant removed and samples air dried 5-10 min. Pellets resuspended in 25 µl milliQ H2O. DNA concentration measured in Qubit with brDNA kit.
ChIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra DNA Library following the manufacturer’s protocol and indexing each sample individually with NEBNext Multiplex Oligos for Illumina.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

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 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 DHRs, the reference genome was broken into 1000 bp windows. Genomic windows with a mean of less than 10 mapped reads per sample were removed prior to further analysis. The MEDIPS R package was then used to calculate differential coverage between control and exposure sample groups as well as disease and non-disease 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 an arbitrarily selected threshold were considered DHRs. The DHR edges were extended until no genomic window with a p-value less than 0.1 remained within 1000 bp of the DHR.
CpG density and other information was then calculated for the 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.

Submission date

Aug 20, 2020

Last update date

Nov 19, 2020

Contact name

Michael K Skinner

E-mail(s)

[email protected]

Organization name

WSU

Department

SBS