GSM5948927: DC5; 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. 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 DNA extraction. Somatic cells and debris were removed by brief sonication (Fisher Sonic Dismembrator, model 300, power 25), then centrifugation and washing 1-2 times in 1X PBS. The resulting pellet was resuspended in 820 µL DNA extraction buffer and 80 µl 0.1M DTT added, then incubated at 65°C for 15 minutes. 80 µl proteinase K (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 rpm 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. After incubation, the sample was spun at 13,500 x 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. 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 10min 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 on a rotator 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 50μl of beads were added to the 500μl of DNA-antibody mixture from the overnight incubation, then incubated for 2h on a rotator 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 250μl digestion buffer (5mM Tris PH 8, 10.mM EDTA, 0.5% SDS) with 3.5μl Proteinase K (20mg/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 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol in -20˚C freezer for one to several hours. The DNA precipitate was pelleted, washed with 70% ethanol, then dried and resuspended in 20μl H2O or 1X TE. DNA concentration was measured in Qubit (Life Technologies) with the ssDNA kit (Molecular Probes Q10212). 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 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. The samples were sequenced on the Illumina HiSeq 2500 at PE50, with a read size of approximately 50 bp and approximately 20 million reads per pool. Twelve libraries were run in one lane.