title: GSM3439708: cauda.d3 DDT; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: Pregnant rats were euthanized when fetuses reached embryonic day 16 (E16) of gestation, and fetal gonads were collected for germ cell preparations. In addition, postnatal day 10 (P10) testes were obtained and used for spermatogonia cell isolation. At E16, sex was determined on the basis of gonadal morphology as previously described (30).  Germ cells were isolated exclusively from males.  Purified populations of male type T1 prospermatogonia (at E16) or postnatal day 10 (P10) spermatogonia were prepared using a mini StaPut gradient method as previously described (30, 43, 44). Briefly, fetal (E16) or postnatal (P10) testes were pooled and dissociated by incubation in 0.25% trypsin-EDTA (Sigma) with vigorous pipetting using a 1000 microliter pipette tip, and the resulting cell solution was filtered through 100 micron nylon mesh to yield a single cell suspension. This cell suspension was then loaded onto a 50 ml 2-4% bovine serum albumen (BSA) gradient prepared in KREBS buffer, and the cells were allowed to sediment at unit gravity at 4˚C for two hours as described (43, 44). The gradient was then fractionated and aliquots of the fractions were examined under phase optics to identify those enriched for the appropriate prospermatogonial or spermatogonial cell types on the basis of morphological characteristics. The enriched fractions were pooled to yield the final sample which was >85% pure for the desired male germ cell type in each case. The pooled samples were then stored at -80°C prior to processing for DNA preparations.  The epididymis was dissected free of connective tissue, a small cut made to the cauda and tissue placed in 5 ml of 1x PBS solution for up to 24 hours at 4°C.  The epididymal tissue was minced and the released sperm was centrifuged at 6,000 x g, then the supernant removed, and the pellet resuspended in NIM buffer, to be stored at -80O C until further use. One hundred µl of sperm suspension was sonicated, spun down at 6,000 g, the pellet washed with 1x PBS once, and then combined with 820 µL DNA extraction buffer and 80 µl 0.1M DTT. The sample was incubated at 65ºC for 15 minutes. Following this incubation 80 µl proteinase K (20 mg/ml) was added and the sample incubated at 55ºC for at least 2 hours under constant rotation. Then 300 µl of protein precipitation solution (Promega, A7953) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 14,000 rpm for 30 minutes at 4ºC. One ml of the supernatant was transferred to a 2 ml tube and 2 µl of glycoblue and 1 ml of cold 100 % isopropanol were added. The sample was mixed well by inverting the tube several times then left in -20ºC freezer for at least one hour. After precipitation the sample was centrifuged at 14,000 rpm for 20 min at 4ºC. The supernatant was taken off and discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol by adding 500 µl of 70% ethanol to the pellet and returning the tube to the freezer for 20 minutes. After the incubation the tube was centrifuged for 10 min at 4ºC at 14,000 rpm and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and then as much liquid as possible was removed with gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 minutes). Pellet was then resuspended in 100 µl of nuclease free water. Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: rat sperm DNA pools were generated using the appropriate amount of genomic DNA from each individual for 3 pools each of control and vinclozolin lineage animals. Genomic DNA was sonicated using the Covaris M220 the following way: the pooled genomic DNA was diluted to 130 μl with TE buffer into the appropriate Covaris tube. Covaris was set to 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 sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube and the volume measured. The sonicated DNA was then diluted with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) to 400 μl, heat-denatured for 10 min at 95˚C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4˚C.   The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 mL 1XPBS with 0.1% BSA and 2mM EDTA) was added and the bead sample was resuspended. Tube was then placed into a magnetic rack for 1-2 minutes and the supernatant discarded. The tube was removed from the magnetic rack and the beads washed once. The washed beads were resuspended in the same volume of 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 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 the incubation the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into magnetic rack for 1-2 minutes and the supernatant discarded, then washed with 1xIP buffer 3 times. The washed bead-DNA solution is then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/ml). The sample was then incubated for 2-3 hours on a rotator at 55˚ C and then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the supe and the tube vortexed for 30 sec then centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20˚C freezer for 1 hour to overnight.  The precipitate was centrifuged at 14,000rpm for 20min at 4˚C and the supernatant removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20˚C freezer for 15 min. then centrifuged again at 14,000rpm for 5min at 4˚C and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and as much liquid as possible was removed with gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 minutes) then resuspended in 20μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP pools 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. NGS was performed by the WSU Spokane Genomics Core using the Illumina HiSeq 2500 with a PE50 application.
attributes.tag: GEO Accession
attributes.value: GSM3439708