UBS-seq of mES DNA
200 ng of mESC gDNA supplemented with 0.1% (w:w) -DNA were fragmented and purified with a DNA Clean & Concentrate Kit (Zymo Research). The fragmented mESC gDNA/-DNA mixture was supplemented with 0.01% (w:w) 200 bp dsDNA oligo and 0.01% (w:w) 164 bp dsDNA oligo as spike-ins. The DNA mixture was then added to reagents from the NEBNext Ultra II End Repair/dA-Tailing Module (NEB) and incubated sequentially at 20 C for 30 mins and 65 C for 30 mins. Using the NEBNext Ultra II Ligation Module (NEB), 1/4 of the DNA mixture, which serves as the UBS-seq control samples, was ligated with 15 M methylated NEBNext adaptor (NEB), while the rest was ligated with 15 M customized DME dsDNA adaptor with phosphorothioate modifications (IDT, Table S1) at 20 C for 15 mins. The ligated DNA samples were purified with 1.0 AMPure XP beads and eluted in H2O. The UBS-seq sample was subjected to UBS-1 conditions and desulphonated via DNA Methylation-Gold Kit (Zymo Research) following the UBS-seq procedure [28].