Genome-wide DNA methylation profiling of mouse embryonic stem cells

WGBS of Atf7ip and Zmym2 KO mESCs was performed as described previously^{64 (link)}. In short, 10 μg genomic DNA was sonicated to a length of approximately 400–500 bp. For each sample, 2 μg sheared genomic DNA was mixed with 10 ng equimolar pooled sonicated methylated phage T7 and unmethylated phage Lambda DNA. Adapter-ligation was carried out with the NEBNext Ultra II kit (NEB E7645L) using methylated adaptors (NEB, E7535S), before bisulfite conversion using the Qiagen Epitect bisulfite conversion kit, according to the manufacturer’s instructions. After conversion, libraries were amplified for 10 cycles using the Pfu TurboCx Hotstart DNA polymerase (Agilent) and the NEB dual index primers (NEB, E7600S). PCR reactions were run with the following parameters: 95 °C for 2 min, 98 °C for 30 s, followed by 10 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 3 min, ending with 5 min at 72 °C. The PCR reactions were cleaned up using 1.2× AMPure XP beads (Beckman Coulter) and eluted in 20 μl EB buffer (Qiagen). Library quality was checked on an Agilent TapeStation and sequencing was done on an Illumina NovaSeq 6000 machine.