GSM6451135: C1; Rattus norvegicus; MeDIP-Seq
For sperm collection, 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 250 µL NIM buffer and stored at -80ºC for intracytoplasmic sperm injection or further processing. Adult female SD rats at 6-12 weeks of age with body weight > 125 grams were used as egg donors. These females were superovulated by intraperitoneal injection of 300 IU/kg of Pregnant Mare's Serum Gonadotropin (PMSG), followed by intraperitoneal injection of 300 IU/kg of human Chorionic Gonadotropin (hCG) 48 h later. Mature oocytes (MII stage) were collected from the oviducts 16 h after hCG injection, and freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma, Cat# H3506) in the M2 medium (Millipore, Cat# MR-015-D) at 37°C for 5 min.  The cumulus-free oocytes were washed and kept in the KSOM-AA medium (Millipore, MR-121-D) in an incubator (Sanyo, Cat# 19AIC) at 37°C with air containing 5% CO2 before ICSI.   For ICSI, sperm frozen in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0) were thawed at 37°C for 3 min. An aliquot of 2 µl sperm TE suspension was mixed immediately with 50 µl of 4% PVP (Sigma, Cat# P5288) in water (Millipore, Cat# TMS-006-C).  A single sperm head was picked up and injected into the mature oocytes using a glass pipette equipped with a piezo drill under the control of an electric micromanipulator (TransferMan NK2, Eppendorf). Injected oocytes were then transferred to the KSOM+AA medium (Millipore, Cat# MR-121-D) covered by mineral oil and cultured in an incubator at 37°C with humidified air containing 5% CO2. After 24 h of culture in KSOM+AA, embryos were transferred into mR1ECM (Cosmo, CSR-R-M191) for further incubation. Morula stage embryos were collected at 100-104 h post-ICSI and into cryovials (Olympus, Cat#: 27-125), which were snap-frozen in liquid nitrogen followed by storage at -80C until shipment on dry ice for MeDIP-seq. Embryos development were observed at 100-104h and 116-120h after ICSI, and morula stage embryos were collected into cryovials (Olympus, Cat#: 27-125), which were snap-frozen in liquid nitrogen followed by storage at -80C until shipment on dry ice for MeDIP-seq. For molecular analysis, an appropriate amount of rat sperm suspension (approximately 50 µL) was used for DNA extraction. 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. 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 the MeDIP, 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 ?l, 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 ?l of 5X IP buffer and 5?g 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 ?l of beads were added to the 500 ?l 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 250 ?l digestion buffer (5mM Tris PH 8, 10.mM EDTA, 0.5% SDS) with 3.5 ?l 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 ?l of GlycoBlue (20 mg/ml), 20 ?l of 5M NaCl and 500 ?l 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 ?l H2O or 1X TE. 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 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.