title: GSM2545061: F1V2; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: The epididymis was dissected free of connective tissue, a small cut made to the cauda and placed in 5 ml of F12 culture medium containing 0.1% bovine serum albumin 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 stored in fresh NIM buffer at -20C until processed further. One hundred ul of human sperm suspension were used, then 820uL DNA extraction buffer and 80ul 0.1M DTT added. The sample was incubated at 65C for 15 minutes. Following the incubation 80 ul proteinase K (20 mg/ml) were added and the sample was incubated at 55C for 2 hours under constant rotation. Then 300 ul 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 20 minutes at 4C. One ml of the supernatant was transferred to a 2 ml tube and 2 ul 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 -20C freezer for at least one hour. After precipitation the sample was centrifuged at 13,500 rpm for 20 min at 4C. The supernatant was taken off and discarded without disturbing the (blue) pellet. The pellet was washed with 70% cold ethanol by adding 500ul 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 4C at 13,500 rpm and the supernatant discarded. The 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 ul of nuclease free water. Equal amounts of DNA from three individual sperm samples were used to produce three DNA pools per lineage and employed for methylated DNA immunoprecipitation (MeDIP). The MeDIP pools were 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. After this step the manufacturer’s protocol was followed.