GSM7821633: P44; Rattus norvegicus; MeDIP-Seq
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. The tissue was then minced, the released sperm pelleted at 4ºC by centrifuging at 3,000 x g for 10 min, then resuspended in 250 µL NIM buffer and stored at -80ºC. For molecular analysis, an appropriate amount of rat sperm suspension (approximately 50 µL) was used for DNA extraction. Previous studies have shown mammalian sperm heads are resistant to sonication unlike somatic cells. Somatic cell contamination and debris were removed by brief sonication (Fisher Sonic Dismembrator, model 300, power level 25), which destroys the somatic cells, then centrifugation and washing 1-2 times in 1X PBS. The resulting purified sperm pellet was resuspended in 820 µL DNA extraction buffer and 80 µl 0.1M DTT added, then incubated at 65°C for 15 minutes. Proteinase K (80 µl of 20 mg/ml) was added and the sample was incubated at 55°C for 2-3 hours under constant rotation. Protein was removed by addition of protein precipitation solution (300 µl, Promega A795A), incubation for 15 min on ice, then centrifugation at 13,500  g for 30 minutes at 4°C. One ml of the supernatant was precipitated with 2 µl of GlycoBlue (Invitrogen, AM9516) and 1 ml of cold 100% isopropanol at -20° overnight. After incubation, the sample was spun at 13,500  g for 30 min at 4°C, then washed with 70% cold ethanol. The pellet was air-dried for about 5 minutes then resuspended in 100 µl of nuclease free water.  For all generations equal amounts of DNA from each individual's sample was used to produce 6 different DNA pools per lineage and the pooled DNA used for methylated DNA immunoprecipitation (MeDIP).  Each pool containted 10-11 individual rat samples for each treatment group, with the exception of one control pool that only contained 5 individual samples. For the MeDIP procedure, genomic DNA was sonicated and run on 1.5% agarose gel for fragment size verification. The sonicated DNA was then diluted with 1X TE buffer to 400 μl, then heat-denatured for 10 min at 95˚C, and immediately cooled on ice for 10 min to create single-stranded DNA fragments. Then 100 μl of 5X IP buffer and 5μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added, and the mixture was incubated overnight with rotation at 4˚C. The following day magnetic beads (Dynabeads M280 Sheep anti-Mouse IgG; Life Technologies 11201D) were pre-washed per manufacturer's instructions, and for each sample 50 μl of beads were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2h with rotation at 4˚C. After this incubation, the samples were washed three times with 1X IP buffer using a magnetic rack. The washed samples were then resuspended in 250 μl digestion buffer (5mM Tris PH 8, 10.mM EDTA, 0.5% SDS) with 3.5 μl Proteinase K (20 mg/ml), and incubated for 2-3 hours on a rotator at 55˚C. DNA clean-up was performed using a Phenol-Chloroform-Isoamyl-Alcohol extraction, and the supernatant precipitated with 2 μl of GlycoBlue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol at -20˚C for one to several hours. The DNA precipitate was pelleted at 13,500  g for 30 minutes at 4°C, washed twice with 70% ethanol, then dried and resuspended in 20 μl H2O. DNA concentration was measured in a Qubit apparatus (Life Technologies) with the ssDNA analysis kit (Molecular Probes Q10212). MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra II RNA Library Prep Kit for Illumina (E770L, New England Biolabs, San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA from the single-stranded DNA resulting from MeDIP. After this step, the manufacturer's protocol was followed indexing each sample individually with NEBNext Multiplex Oligos for Illumina.