GSM1973810: HF33; Mus musculus; Bisulfite-Seq
Cauda epididymis and vas deferens was rapidly dissected and punctured, and was incubated in 500 ¼L of human tubule fluid (HTF - Millipore MR-070-D) at 37 C for 30 minutes. Supernatant was removed, sperm were pelleted at 2000 g for 5 minutes, washed once with 1 mL water and then pelleted. Sperm were washed again with 1 mL PBS, and pelleted again. Sperm were resuspended in 400 ¼L DNA Lysis Buffer (10mM Tris, 5mM EDTA, 0.5% SDS, and 200mM NaCl) with 10mM DTT, and incubated at 37oC for 30 minutes. Sperm were subjected to needle homogenization. 20 ¼g/mL Proteinase K was added to the homogenate and incubated at 55oC for 16 hours.  DNA was extracted with Phenol:Chloroform:Isoamyl Alcohol and precipitated with 100% EtOH. DNA was sheared to an average length of ~100-500 bp with a Covaris sonicator, fragment ends were cleaned up and A-tailed. Fragments were ligated to pre-methylated Illumina paired-end adaptors, bisulfite converted, and PCR-amplified.