Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen). AbaSeq libraries for 5hmC profiling were constructed as previously described15 (link). In brief, genomic DNA was glucosylated, then digested by AbaSI enzyme (NEB). Biotinylated P1 adapters were ligated onto the AbaSI digested DNA then fragmented using a Covaris S2 sonicator (Covaris), following the manufacturer’s instructions. Sheared P1-ligated DNA was then captured by mixing with Dynabeads MyOne Streptavidin C1 beads (Life Technologies) according to the manufacturer’s specifications. End repair and dA-tailing were carried out on the beads by using the NEBNext End Repair Module (NEB) and the NEBNext dA-tailing Module (NEB) at 20°C and 37 °C respectively for 30 min. P2 adapters were ligated to the random sheared ends of the dA-tailed DNA. Finally, the entire DNA was amplified using the Phusion DNA polymerase (NEB) with the addition of 300 nM forward primer (PCR_I) and 300 nM reverse primers (PCR_IIpe) for 16 cycles. The libraries were purified using AMPure XP beads (Beckman-Coulter) and sequenced on the Illumina HiSeq 2000 instrument.