For the ACE-seq libraries construction, 200 ng of the genomic DNA was firstly fragmented into 300 bp by the Covaris M220. Then, the T4 Phage β-glucosyltransferase (NEB) and uridine diphosphoglucose were used to convert 5hmC into 5ghmC at 37 °C for 2 h. After that, 0.1 M NaOH (5×) was added to the modified fragments for denaturation at 50 °C for 10 min. Then, the DEA enzyme mix (Vazyme) was used to deaminate dC or 5mC to U or T, respectively, at 37 °C for 3 h. After deamination, DNA fragments were purified and subjected to library construction with Scale Methyl-DNA Lib Prep Kit according to the instruction manual (Illumina). Briefly, the single-stranded heat-denatured DNA fragments were ligated to T7 truncated adapter and dA-tailed. Next, the ligated DNA was extended to the second strand synthesis and the methylated adapters were ligated. The DNA was ampliﬁed for 10 cycles using Illumina 8-bp dual index primers. Finally, the ACE-seq libraries were analyzed by Agilent 2100 Bioanalyzer and sequenced on an Illumina NovaSeq 6000 platform by 150 bp paired-end sequencing.