GSM7438686
Cauda epididymis and vas deferens were minced and placed in 37°C warm M2 medium for at least 15 minutes. Sperm cells were separated from contaminating tissue by filtration through a 70 um Nylon mesh using several rinses with PBS. Cells were inspected for impurities under the microscope followed by cell counting and aliquoting. 100 000 fresh PBS washed sperm cells were incubated on ice for 3 minutes in resuspension buffer (0.1% v7v NP-40, 0.1% v/v Tween-2 and 0.01% v/v Digitonin, 10mM Tris-HCL pH7.5, 10mM NaCl, 3mM MgCl2), after a washing step (10mM Tris-HCL pH7.5, 10mM NaCl, 3mM MgCl2) nuclei were recovered by centrifugation (500g, 10 min, 4°C). Nuclei were transposed in TD Buffer supplemented with Tween-20 0.1% v/v, Digitonin 0.01% v/v and 2.5 ul Tn5 Transposase for 30 min at 37°C while shaking (1.000rpm). DNA was purified using the Qiagen MinElute Reaction Cleanup and amplified using the NEBNext high-Fidelity 2X master mix and 2.5uM nextera primers for 72°c for 5 minutes, 98°C for 30 sec and 6 cycles of 98°C for 10 sec and 63§C for 30 sec, followed by 1 minute at 72°C. A subfraction of the amplification product was then subjected to q-PCR to evaluate how many additional PCR cycles would be required. Remaining PCR product was then amplified for additional cycles before double sided bead purification with AmPure XP beads. Libraries were inspected quantitatively and qualitatively on an Agilent Bioanalyzer. Sequencing aimed at 50 Million reads/sample.