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Sample GSM4769606

Query DataSets for GSM4769606

Status

Public on Dec 02, 2020

Title

DX1

Sample type

SRA

Source name

Sperm

Organism

Rattus norvegicus

Characteristics

measurement: MeDIP
disease: kidney disease
lineage: dioxin
cell type: Sperm

Treatment protocol

Outbred Sprague Dawley gestating female rats (F0 generation) were administered an intraperitoneal dose of 100 mg/kg body weight of dioxin (TCDD). The lowest observable adverse effects level (LOAEL) is 160 ng/kg/BW. 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 dioxin 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. DNA protocol: While sperm are thawing, turn on the centrifuge to 4ºC. 1. Re-suspend sperm pellet with pestle (and pestle motor if available). o If there is some liquid left with the pellet, there is no need to add any additional buffer o If not add 100 l 1xPBS or NIM buffer 2. Take 25 to 50 l of the suspension (depending on how big the pellet is) and transfer it to a microfuge tube with 200 l 1xPBS. o Mix 3. Sonicate suspension at 25% with probe sonicator for 5 seconds. 4. Spin at 12,000 rpm for 10 minutes at 4C. o While waiting, turn on heat plate to 65C 5. Discard supernatant. Be careful – pellet is loose. 6. Wash with 200 l 1xPBS and spin as above. o While waiting, retrieve DTT from freezer (make sure there is enough) o Turn on the incubator o Vortex DTT once it has thawed a bit 7. Resuspend pellet in 100 l 1xPBS, then continue with protocol below. General: 1. Re-suspend tissue in 100 l PBS or NIM buffer, then o add 820 l DNA extraction buffer o add 50 l 0.1M DTT o vortex briefly o incubate at 65C for 15 minutes o Retrieve ProteinaseK from freezer 2. Add 50 l proteinase K (20 mg/ml) and o incubate at 55C for 2-3 hours under constant rotation o use Eppendorf SafeLock tubes and wrap the tops with Parafilm 3. After incubation add 300 l of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) o mix (vortex) thoroughly and make sure that the solutions are mixed o then incubate for 20 min on ice (or longer if needed) 4. Centrifuge at 13,500 g for 30 minutes at 4C. o Prepare 2 mL tubes o Take glycoblue out of the freezer 5. Transfer 1 ml of the supernatant to a new 2 ml tube. o add 2 l of glycoblue o add 1 ml of cold 100 % isopropanol 6. Mix well by inverting tubes several times. 7. Incubate for 60 min at -20C. 8. Centrifuge for 20 min at 13,500 g at 4C. 9. Take off and discard supernatant without disturbing (blue) pellet. 10. Wash pellet 2-3 times with 70% cold ethanol. o Add 500l of 70% ethanol to each pellet and o return to freezer for 20 minutes 11. Centrifuge for 10 min at 4C at maximum speed (18,200 g) o discard supernatant 12. Spin again briefly to collect residual ethanol at bottom of tube and remove as much liquid as possible with gel loading tip. 13. Air-dry at RT until pellet looks dry, but be careful not to over-dry. o This will take about 5 minutes 14. Re-suspend in 100 l of nuclease free water or TE. 15. Measure DNA concentration using Nanodrop. Methylated DNA immunoprecipitation protocol: Shear & Add Antibody to DNA 1. Sonicate purified genomic DNA using Covaris: a. Dilute 6μg genomic DNA into 50 or 130 μl TE buffer in the appropriate Covaris tube. b. Set Covaris to desired program (300 or 500 bp program works well). c. Run program for each tube • While waiting, turn on the dry bath to 95ºC. 2. Run 10 μl sonicated DNA & 5 μl DNA ladder on 2% agarose gel to verify fragment size. • While waiting for gel, clean up Covaris. 3. Measure volume of sonicated DNA after gel run. 4. Dilute sonicated DNA with TE buffer to 400 μl (400 – 130 + 10 = 280 µL) 5. Heat-denature for 10min at 95˚C, and • immediately cool on (watery) ice for 5min 6. To the denatured sonicated DNA: a. Add 100μl of cold 5X IP buffer b. Add 5μg (volume depending on concentration) of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006). ALWAYS check antibody concentration. Diagenode sometimes changes it. c. Incubate the DNA-antibody mixture overnight on a rotator at 4˚C. Rotate at a low enough speed to prevent significant foaming. Bind Beads to DNA: Antibody Mixture 1. Pre-wash Magnetic Beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) as follows: • Resuspend the beads in the vial (pipet up and down or rotate, do not vortex) • Transfer desired volume (50 μl per sample) to a centrifuge tube, add the same volume of Washing Buffer (at least 1 mL), and resuspend. • Place the tube in a magnet for 1-2 minutes and discard supernatant. • Resuspend beads in 1 ml of washing buffer and incubate for 1 minute on bench. Put tube on magnet for 1-2 minutes and discard supernatant. • Remove the tube from the magnet and resuspend the washed beads in the same volume of 1xIP buffer as the initial volume of beads. 2. Add 50μl of beads to the 500μl of DNA-antibody mixture from step 2 3. Incubate 2h on a rotating platform at 4˚C. Wash DNA: Antibody: Beads Mixture & Add Proteinase K Mix 1. Wash beads three times with 1X IP buffer as follows: • Place tube in magnet for 1-2 minutes and discard supernatant • Remove tube from magnet • Add 1ml cold 1X IP buffer • Mix by inverting tube or gently vortexing (set Vortex to low). • Incubate tube(s) for 1 minute on bench. • Place tube in magnet for 1-2 minutes and discard supernatant • Repeat twice for a total of 3 washes 2. Resuspend the beads in 250μl digestion buffer. 3. Add 3.5μl Proteinase K Mix (20mg/ml) to the resuspended beads. 4. Incubate 2-3 hours on a rotating platform at 55˚C (make sure to seal lids so they don’t leak). Use USA Scientific SealRite tubes or Eppendorf Safe-Lock tubes and seal top with Parafilm. Purify DNA • Retrieve Glycoblue from freezer. 1. Remove Parafilm and add 250μl Phenol-Chloroform-Isoamylalcohol to each tube. • Vortex for 30 sec and centrifuge at 14,000g for 5min at RT. • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube. 2. Add 250μl chloroform to the supernatant from step 1. • Vortex for 30sec and centrifuge at 14,000g for 5min at RT. • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube. 3. Add 500 μl ethanol • Then add 20μl 5M NaCl • Then add 2μl glyco-blue (20mg/ml) • Vortex well 4. Precipitate in -20˚C freezer for 1 hour to overnight. 5. Centrifuge at 14,000g for 20min at 4˚C. Carefully remove the supernatant, while not disturbing the pellet. 6. Wash the pellet with 500μl cold 75% ethanol. Leave in -20˚C freezer for 15 min. 7. Centrifuge at 14,000g for 5min at 4˚C. Carefully remove the supernatant. 8. Completely dry samples. 9. Resuspend in 25μl H2O. 10. Measure the DNA concentration in Qubit with ssDNA kit and matching buffer.
MeDIP 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. NEBNext Ultra II Library Prep Kit for Illumina: ssDNA • Start with 20 µl in PCR strips. o ~40 ng of DNA o Complete the volume to 20 µl • Add 2.5 µL of diluted (1:10) random primer to each tube. o for 10 samples, 27 µL 0.1x TE and 3 µL primers o for 12 samples 36 µL 0.1x TE and 4 µL primers o for 22 samples, 54 µL 0.1x TE and 6 µL primers • Place strips in the thermocycler and use the Denaturation program. Immediately remove (as soon as time runs out on the program) and cool on watery ice following denaturation. Let tubes rest on watery ice for 5 minutes, then transfer to refrigerated block. I. Second Strand cDNA Synthesis Assemble the second strand cDNA synthesis reaction on ice by adding the following components to the 20 µl DNA. Second Strand Synthesis Reaction DNA 20 µL ORANGE NEBNet 2nd Strand Synthesis Reaction Buffer 8 µL ORANGE NEBNext 2nd Strand Synthesis Enzyme Mix 4 µL Nuclease-free water 45.5 µL Total Volume

Library strategy

MeDIP-Seq

Library source

genomic

Library selection

5-methylcytidine antibody

Instrument model

Illumina HiSeq 2500

Description

kidney.dmr.results.csv.gz

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 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, 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 disease and non-disease 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 an arbitrarily selected threshold were considered DMRs. The DMR edges were extended until no genomic window with a p-value less than 0.1 remained within 1000 bp of the DMR.
CpG density and other information was then calculated for the DMR 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

Sep 04, 2020

Last update date

Dec 02, 2020

Contact name

Michael K Skinner

E-mail(s)

skinner@mail.wsu.edu

Organization name

WSU

Department

SBS