GSM6705599: IC2P1; Rattus norvegicus; MeDIP-Seq
The epididymis was dissected free of fat and connective tissues, then, after cutting open the cauda, placed into 6 mL of phosphate buffer saline (PBS) for 20 min at room temperature. Further incubation at 4 ºC will immobilize the sperm. The tissue was then minced, the released sperm was pelleted at 4 ºC 3000 x g for 10 min, then resuspended in NIM buffer and stored at -80 ºC 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 0.1 M DTT added, then incubated at 65 °C for 15 min. Eighty μL proteinase K (20 mg/mL) was added and the sample was incubated at 55 °C for 2−3 h 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 x g for 30 min 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 min then resuspended in 100 μL of nuclease free water. For the MeDIP procedure, F1 generation samples were pooled, with three to four samples per pool and six pools each for both the ifosfamide and control lineage. F2 generation samples were pooled as well, with seven samples per pool for the control lineage, and eight or nine samples per poor for the ifosfamide lineage. 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 5 μg 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 50 μl of beads were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 h 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 250 μl digestion buffer (5 mM Tris PH 8, 10.mM EDTA, 0.5 % SDS) with 3.5 μl Proteinase K (20 mg/mL), and incubated for 2−3 h 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 5 M NaCl and 500 μl ethanol in -20 °C freezer for one to several hours. The DNA precipitate was pelleted, washed with 70 % ethanol, then dried and resuspended in 20 μl 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.