Imprint modification kit

Total DNA from FACS-sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and DNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 26 µL final volume using the Savant SpeedVac Concentrator (Thermo) and following the manufacturer’s instructions. Genomic DNA was digested by 20 units of MspI enzyme (NEB) in NEB buffer 2 at 37°C for 3 hrs, and digested DNA was purified using AMPure XP beads (Beckman-Coulter). Libraries were made following the NEBNext Ultra DNA Library Prep protocol with methylated adaptors and the following modifications: following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma); and PCR enrichment was carried out for 18 cycles using the KAPA Uracil⁺ DNA polymerase master mix (KAPA Biosystems) and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Pooled libraries were sequenced on the Illumina HiSeq 2500 instrument, using the 'dark sequencing' protocol, as previously described32 (link).

Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen). In some cases, unmethylated λ phage DNA (Promega) was spiked in following DNA isolation to assess bisulphite conversion rate. DNA was fragmented using a Covaris S2 sonicator (Covaris), as per manufacturer’s instructions. Libraries were made following the NEBNext Library Prep protocol, with methylated adaptors and the following modifications: following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma); and PCR enrichment was carried out for 16 cycles using the NEXTflex Bisulphite-Seq Kit for Illumina Sequencing (Bioo Scientific) master mix and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Libraries were sequenced on the Illumina HiSeq 2000 or 2500 instrument.