title: GSM3324498: sc.v3: Sertoli Cells Vinclozolin; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: The 20-day-old rats were sacrificed and the testes dissected. Testes were washed two times with HBSS. The tunica albuginia was removed from each testis and discarded. The tissue was then chopped with a razor blade until it appeared homogeneous and then underwent sequential enzymatic digestion and wash steps to isolate the purified Sertoli cells. The first step uses trypsin (0.25%) and DNase (0.3 mg/ml) followed by a trypsin inhibitor (0.3 mg/ml) incubation and washes. The next digestion step involves collagenase (0.5 mg/ml) and DNase followed by the final digestion with hyaluronidase (1 mg/ml) and DNase. The final pellet will contain the purified Sertoli cells. DNase I (Sigma DN-25), Collagenase (Sigma C1639), Hyaluronidase (Sigma #H3757), Trypsin Inhibitor Type I-S (Sigma T-6522). Sertoli cell pellets were resuspended in 100 μL 1xPBS, then combined with 820 μL DNA extraction buffer (50 mM, Tris HCl, 10 mM EDTA, pH 8.0, 0.5% SDS) and 80 μl proteinase K (20 mg/ml). The sample was 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 and 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: DNA isolated from Sertoli cell pools (3 pools each of control, DDT and vinclozolin lineage animals) was sonicated using the Covaris M220 the following way: the 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 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.
attributes.tag: GEO Accession
attributes.value: GSM3324498