GSM2359642: F1P4; Danio rerio; MeDIP-Seq
One hundred μl of sperm suspension was used then 820 μl DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl 0.1 M Dithiothreitol (DTT) added and the sample incubated at 65 oC for 15 minutes. Proteinase K (80 μl, 20 mg/ml) was added and the sample incubated on a rotator at 55 oC for 2 hours. After incubation, 300 μl of protein precipitation solution (Promega, A795A, Madison, WI) was added, the sample mixed and incubated on ice for 15 minutes, then spun at 4 oC at 13,000 rpm for 20 minutes. The supernatant was transferred to a fresh tube, then precipitated over night with the same volume 100% isopropanol and 2 μl glycoblue at -20 oC. The sample was then centrifuged and the pellet washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop. Zebrafish sperm DNA pools were generated using 2 μg of genomic DNA from each individual for 3 pools each of control and MeHg exposed animals. Each pool contained 2 individuals for a total of n=6 per exposure group. The resulting 6 μg of genomic DNA per pool was diluted to 150 μl with 1x Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with a probe sonicator using 5 x 20 pulses at 20% amplitude. Fragment size (200-800 bp) was verified on a 1.5% agarose gel. Sonicated DNA was diluted to 400 μl with 1xTE and heated to 95 oC for 10 minutes, then incubated in ice water for 10 minutes. Then 100 μl of 5 x immunoprecipitation (IP) buffer (50 mM Sodium Phosphate pH 7, 700 mM NaCl, 0.25% Triton X-100) and 5 μg of 5-mC monoclonal antibody (Diagenode, Denville, NJ, C15200006-500) were added and the sample incubated on a rotator at 4 oC overnight. The next day Protein A/G Agarose Beads from Santa Cruz were prewashed with 1xPBS/0.1% BSA and resuspended in 1 x IP buffer. Eighty μl of the bead slurry were added to each sample and incubated at 4 oC for 2 hours on a rotator. The bead-DNA-antibody complex was washed 3 times with 1 x IP buffer by centrifuging at 6,000 rpm for 2 minutes and resuspending in 1 x IP buffer. After the last wash the bead-complex was resuspended in 250 μl of digestion buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) with 3.5 μl Proteinase K (20mg/ml) per sample and incubated on a rotator at 55 oC for 2 hours. After incubation, DNA was extracted with the same volume of Phenol-Chloroform-Isoamyalcohol and then with the same volume chloroform. To the supernatant from chloroform extraction 2 μl glycoblue, 20 μl 5M Sodium Chloride and 500 μl 100% cold ethanol were added.   DNA was precipitated at -20 oC overnight, then spun for 20 minutes at 13,000 rpm at 4 oC, washed with 75% ethanol and air-dried. Dry pellet was resuspended in 20 μl H2O and concentration measured in Qubit using the Qubit ssDNA Assay Kit The MeDIP pools were used to create libraries for next generation sequencing (NGS) at the University of Reno, NV, Genomics Core Laboratory 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. After this step the manufacturer’s protocol was followed. Each pool received a separate index primer. NGS was performed at that same laboratory using the Illumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 100 million reads per pool.
GEO Accession
GSM2359642