GSM3326033: gran.d1; Rattus norvegicus; MeDIP-Seq
F3 generation rats from vinclozolin, DDT and control lineages were treated with Pregnant Mare Serum Gonadotropin (Sigma cat, St. Louis, MO) (10 IU PMSG injected IP) at 20-22 days of age. Two days later animals were sacrificed and ovaries removed. The ovarian bursa and its adherent fat were removed from each ovary and the ovaries processed for granulosa cell collection. The ovaries were suspended in the Ham's F-12 base medium used for all procedures (Thermo Scientific). Following sequential 30 minute incubations at 37 °C in 6 mM EGTA in F-12 (to decrease Ca2+ - mediated cell adhesion) and then 0.5 M sucrose in F-12 (to increase osmotic pressure within follicles), ovaries were returned to F-12. Granulosa cells were released into the medium from antral follicles using 30-gauge needles and gentle pressure. Oocytes were removed by aspiration under a dissecting microscope. Granulosa cells from 4-9 rats from the same treatment group were pooled and collected into 1.5 ml tubes, allowed to settle for 10 minutes and supernatant discarded. Three pools of granulosa cells were prepared from different animals and ovaries for each treatment group.  Samples were stored at −70° until the time of DNA isolation. A DNA sample was mixed with 820 μl DNA extraction buffer and 80 μl 0.1M DTT (dichlorodiphenyltrichloroethane) added. The sample was incubated at 65°C for 15 minutes. Following this incubation 80 μl proteinase K (20 mg/ml) was added and the sample was incubated at 55°C for 2 hours under constant rotation. Then 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) were added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 17,000xg for 20 minutes at 4°C. One ml of the supernatant was transferred to a 2 ml tube and 2 μl of Glycoblue (Thermo-Fisher AM9515) and 1 ml of cold 100 % isopropanol were added. The sample was mixed well by inverting the tube several times then left in -20°C freezer for at least one hour. After precipitation, the sample was centrifuged at 17,000xg for 20 min at 4°C. The supernatant was taken off and discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol and incubated at -20°C for 20 minutes. Samples were centrifuged for 10 min at 4°C at 17,000xg and the supernatant discarded. The pellet was air-dried at room temperature (about 5 minutes). The pellet was then resuspended in 100 μl of nuclease free water. Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed. The genomic DNA was sonicated using the Covaris M220 the following way: up to 6μg of pooled granulosa cell genomic DNA was diluted to 130 μl with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) into a Covaris tube. The Covaris was set to the 300 bp program and the program was run for each tube in the experiment. 10 μl of each sonicated DNA was run on 1.5% agarose gel to verify fragment size. The remaining DNA was diluted with TE buffer to 400 μl, heat-denatured for 10min at 95˚C, then immediately cooled on ice for 10 min. Then 100μl of 5X IP buffer and 5μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added, and the DNA-antibody mixture was incubated overnight with rotation at 4˚C.   The following day 50μl of pre-washed anti-mouse magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; Life Technologies 11201D) were added to the DNA-antibody mixture, then incubated for 2h on a rotator at 4˚C.  Then the DNA-antibody-bead mixture was placed into a magnetic rack for 1-2 minutes and the supernatant discarded, then washed with 1xIP buffer 3 times. The washed bead mixture is then resuspended in 250μl digestion buffer (5mM Tris PH8, 10.mM EDT4, 0.5% SDS with 3.5μl Proteinase K (20mg/ml)). The sample was then incubated for 2-3 hours on a rotator at 55˚. 250μl of buffered Phenol-Chloroform-Isoamylalcohol solution were added to the sample and the tube vortexed for 30 sec then centrifuged at 17,000xg for 5min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250μl chloroform were added to the supernatant from the previous step, vortexed for 30sec and centrifuged at 17,000xg for 5min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of Glycoblue (20mg/ml) (Invitrogen AM9516), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated at -20˚C for >1 hour.  The DNA precipitate was centrifuged at 17,000xg for 20min at 4˚C and the supernatant removed. The pellet was washed with 500μl cold 70% ethanol and incubated at -20˚C for 15 min. then centrifuged again at 17,000xg for 5min at 4˚C and the supernatant discarded. The pellet was air-dried at RT (about 5 minutes) then resuspended in 20μl H2O or TE. DNA concentration was measured using a Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples were used to create libraries for next generation sequencing (NGS) using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each pool received a separate index primer.
GEO Accession
GSM3326033