title: GSM2967081: P1 ChIP-seq; Rattus norvegicus; ChIP-Seq
library_construction_protocol: The epididymis was dissected free of connective tissue, a small cut made to the cauda and tissue placed in 5 ml of 1xPBS for 10 minutes at 37°C and then kept at 4ºC to immobilize the sperm.  The epididymal tissue was minced and the released sperm centrifuged at 13, 000 x g and the pellet stored at -20ºC until processed further. The pellet was resuspended in NIM buffer and then one hundred μl of sperm suspension was combined with 820 μL DNA extraction buffer and 80 μl 0.1M DTT. 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 incubated at 55°C for at least 2 hours under constant rotation. Then 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 13,500 rpm for 20 minutes at 4°C. 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 -20°C freezer for at least one hour. After precipitation the sample was centrifuged at 13,500 rpm 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 by adding 500μl 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 4°C at 13,500 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. Equal amounts of DNA from several (3-6) individual sperm samples were used to produce three DNA pools per lineage and employed for methylated DNA immunoprecipitation (MeDIP). 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 DDT lineage animals. Genomic DNA was sonicated using the Covaris M220 the following way: the pooled genomic DNA was diluted to 130 μl with TE 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 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 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) 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 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 250μl digestion buffer with 3.5μl Proteinase K (20mg/ml). The sample was then incubated for 2-3 hours on a rotator at 55˚ and then 250μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the supe and the tube vortexed for 30 sec then centrifuged at 14,000rpm 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 14,000rpm 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), 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). Histone chromatin immunoprecipitation with genomic DNA was performed as follows: rat sperm pools were generated using a total of 8 million sperm for 3 pools of control and DDT lineage animals. The control pools contained 5-6 individuals for a total of n=17 rats, the DDT pools contained 4 individuals for a total of n=12 rats per exposure group. Sperm from each animal was sonicated 10 seconds using a Fisher Sonic Dismembrator Model 300 then counted individually on a Neubaeuer improved cell prior to pooling. The sperm pools were filled up to 1 ml with 1X PBS. To reduce disulfide bonds, 50 µl of 1M DTT was added to each pool and incubated for 2 hours at room temperature under constant rotation. To quench any residual DTT in the reaction, 120 µl of 1M of NEM (N-Ethylmaleimide, Thermo Scientific) was then added and incubated for 30 min at room temperature under constant rotation. The sperm cells were pelleted at 2,000g for 5 min at room temperature and the supernatant discarded. Pellets were resuspended in 1X PBS. The mixture was spinned again at 2,000g for 5 min at room temperature and the supernatant was discarded. The sperm cells were then resuspended in “complete buffer” in a ratio of 2 million sperm cells in 50 µl (as described by Hisano et al. 2013, (59)). “Complete buffer” was supplemented with tergitol 0.5% and DOC (sodium deoxycholate, Sigma Aldrich 30970). 50 µl of this mix was added to each aliquot. The tubes were homogenized and incubated for 20 min on ice. 10 Kuntz units of MNase (Roche, cat. no. 10107921001) were added and the samples incubated for 5 min at 37ºC. The reaction was stopped by the addition of 2 µl of EDTA 0.5 M. The sperm cells were aliquoted in ~ 4 µg of DNA. The genomic DNA was sonicated using the Covaris M220 the following way: ~ 4 µg of genomic DNA was resuspended in 130 µl of “complete buffer” supplemented with tergitol 0.5% and DOC 1%. Covaris was set to “Chromatin shearing” program for 10 min and the program was run for each tube in the experiment. Whether the samples were treated using the MNase or the Covaris, 10 µl of each sample was run on a 1.5% agarose gel to verify fragment size. The aliquots from the same sample were pooled back together and centrifuged at 12,500 rpm for 10 min at room temperature. The supernatant was transferred to a fresh microfuge tube. 65 µl of protease inhibitors were added in each sample. 3 µl of antibody (monoclonal r