title: GSM3330324: pr.str.c1; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: Ventral prostate epithelial and stromal cells were isolated. Ventral prostates were removed from 19-21 day old rats and cleaned of fat, then digested in 50 ml Hank's Buffered Salt Solution (HBSS) with 0.5 mg/ml collagenase type II (Sigma C1764) and 66μg/ml DNAse (Sigma DN25) with agitation at 37° for 4 hours. After gravity settling for 10 min. the supernatant containing the stromal cells was removed. The supernatant was centrifuged at 30xg for 4 min. to pellet contaminating epithelial cells, and then the supernatant centrifuged again at 190xg for 6 min to pellet the stromal cells. This wash is repeated 1-2 more times. To clean the epithelial cells from the original gravity settled pellet, the pellet is resuspended in HBSS, centrifuged at 30xg for 4 min., and the supernatant discarded. This wash is repeated 1-2 more times. The cell pellet was resuspended in 820 μL DNA extraction buffer (0.05M Tris HCl, pH 8; 0.01M EDTA; 0.5% SDS) and 80 μl proteinase K (20 mg/ml) added. The sample was incubated at 55°C for 2-3 hours under constant rotation. Then 300 μl of protein precipitation solution (Promega A795A) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 17,000xg for 30 minutes at 4°C. One ml of the supernatant was transferred to a 2 ml tube and 2 μl of Glycoblue cryoprecipitant (Thermo Fisher Scientific AM9516) and 1 ml of cold 100% isopropanol were added. The sample was mixed well by inverting the tube several times then incubated at -20°C for at least one hour. After precipitation, the sample was centrifuged at 17,000xg for 20 min at 4°C. The supernatant was discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol and incubated at -20°C for 20 minutes. Samples were centrifuged for 10 min at 4°C at 17,000xg and the supernatant discarded. Pellet was air-dried at RT (about 5 minutes), then resuspended in 100 μl of nuclease free water. The genomic DNA was fragmented using the Covaris M220. 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 remaining DNA was 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, and the DNA-antibody mixture was incubated overnight with rotation at 4˚C.   The following day 50μl of pre-washed anti-mouse IgG magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; Life Technologies 11201D) were added to the DNA-antibody mixture, then incubated for 2h on a rotator at 4˚C. Then the DNA-antibody-bead mixture was placed into a magnetic rack for 1-2 minutes and the supernatant discarded, then the pellet washed with 1xIP buffer 3 times. The washed sample is then resuspended in 250μl digestion buffer (5mM Tris PH8, 10.mM EDT4, 0.5% SDS) with 3.5μl Proteinase K (20mg/ml)) and incubated for 2-3 hours on a rotator at 55˚. 250μl of buffered Phenol-Chloroform-Isoamylalcohol solution were added to the sample and the tube vortexed for 30 sec then centrifuged at 17,000xg for 5min at room temperature. The aqueous supernatant was 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 17,000xg 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) (Invitrogen AM9516), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated at -20˚C for >1 hour.  The DNA precipitate was centrifuged at 17,000xg for 20min at 4˚C and the supernatant removed. The pellet was washed with 500μl cold 70% ethanol and incubated at -20˚C for 15 min. then centrifuged again at 17,000xg for 5min at 4˚C and the supernatant discarded. The pellet was air-dried at RT (about 5 minutes) then resuspended in 20μl H2O or TE. DNA concentration was measured using a 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.