title: GSM3333135: sP24; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: The epididymis from each rat was dissected free of fat and connective tissue, a small cut made to the cauda and the tissue placed in 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature and then kept at 4ºC to immobilize the sperm.  The epididymal tissue was coarsely minced and the released sperm centrifuged at 4ºC 3,000 x g for 10 min and pellet resuspended in fresh NIM buffer and stored at -80ºC until processed further. Twenty–fifty to 50 μl of rat sperm suspension were used for DNA extraction. The suspension was sonicated for five seconds (Fisher Sonic Dismembrator, model 300, power 25), then centrifuged 5 min at 6000 x g, supernatant discarded and sperm washed again to remove somatic cells and debris. Then the pellet was resuspended in 820 μL DNA extraction buffer and 80 μl 0.1M DTT 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 at least 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 13,500 rpm for 30 minutes at 4°C. One ml of the supernatant was transferred to a 2 ml tube and 2 μl of glycoblue (Invitrogen, AM9516) and 1 ml of cold 100 % isopropanol were added. The sample was mixed well by vortexing the tube then left in -20°C freezer for at least one hour. After precipitation, the sample was centrifuged at 13,500 x g for 30 min at 4°C. The supernatant was taken off and discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol, then tube was spun again briefly to collect residual ethanol at 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. For all generations equal amounts of DNA from each individual's sample was used to produce 6 different DNA pools per lineage and the pooled DNA used for methylated DNA immunoprecipitation (MeDIP). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: The genomic DNA was sonicated using the Covaris M220 as follows: 6μg of pooled or individual animal sperm genomic DNA was diluted to 130 μl with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) 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 then 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 to the denatured (single-stranded) 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; Life Technologies 11201D) were pre-washed per manufacturer's instructions and 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-DNA-antibody samples were washed three times with 1X IP buffer using a magnetic rack. The washed samples are then resuspended in 250μl digestion buffer (5mM Tris PH 8, 10.mM EDTA, 0.5% SDS) with 3.5μl Proteinase K (20mg/ml) and incubated for 2-3 hours on a rotator at 55˚. Samples were then extracted with Phenol-Chloroform-Isoamyalcohol and the final supernatant precipitated with 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol in -20˚C freezer for >1 hour to overnight.  The DNA precipitate was centrifuged at 14,000rpm for 20min at 4˚C, the pellet was washed with 500μl cold 70% ethanol and then air-dried at RT (about 5 minutes). It was resuspended in 20μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA was used to create libraries for next generation sequencing (NGS) 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 from the single-stranded DNA resulting from MeDIP. After this step the manufacturer's protocol was followed. Each pool received a separate index primer.
attributes.tag: GEO Accession
attributes.value: GSM3333135