GSM7157111
For sperm cell sorting, mouse cauda epididymides were dissected into a Petri dish and fat patches were removed with forceps and scissors. Each epididymis was punctured with a needle and carefully squeezed into 100 ul of PBS with the help of two forceps. Sperm was transferred into an Eppendorf tube and was allowed to liquefy at room temperature for 5 minutes. To break sperm tails, cells were briefly sonicated with a Brandson Tip digital sonicator with 10% amplitude and 3 cycles of 0.5 sec ON / 2 sec OFF. Cells were stained with 2 ul/ml Hoechst 33342 (H3570, ThermoFisher) for 1h at 25°C with constant shaking and protected from light. Sperm cells were filtered through a 40 μm Nylon filter into 5 ml polypropylene tubes. Sperm cells were sorted for 1C DNA content. Pellets of 1 million sperm cells were incubated in 200μl of DNA extraction buffer (20mM Tris-HCl pH 7.5, 300mM NaCl, 2% SDS, 10mM EDTA, 200 μg/ml Proteinase K) for 16 hours at 55oC. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution was added to the samples. After a vigorous vortexing for 30 seconds and centrifugation at 16.000g for 10 minutes, the upper aqueous phase was transferred to new tubes. 0.05 μg/μl glycogen, 1/10 volume of 3M sodium acetate and 2.5 volumes of 100% ethanol were added, and the samples were incubated fot 2 hours at -80oC. DNA precipitated by centrifugation at 16000g for 30m minutes at 4oC. DNA pellet was washed once with 70% ethanol and resuspended in 20 μl. DNA was further purified using 2 volumes of Ampure XP beads (Beckman Coulter). Genomic DNA (100ng per reaction) was sheared to an average fragment size of 400 bp using the Covaris S220 instrument. The sheared DNA was used as input for the NEBNext Enzymatic Methyl-seq (NEB E7120) following the manufacturer's instructions except for doubling the reaction incubation times. The libraries were amplified for 5 cycles and sequenced in 2x75-bp paired-end mode with NextSeq 500 sequencing technology (Illumina), per the manufacturer's recommendations.