Hiseq x ten sequencer pe150 mode

The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to isolate the high-quality DNA from each sample. According to the manufacturer’s instructions, 1 μg of genomic DNA was fragmented by sonication to a mean size of approximately 250 bp and subsequently used for whole genome bisulfite sequencing (WGBS) library construction using an Acegen Bisulfite-Seq Library Prep Kit (Acegen, Shenzhen, GD, China). Briefly, fragmented DNA was end-repaired, 5′-phosphorylated, 3′-dA-tailed, and then ligated to methylated adapters. The methylated adapter-ligated DNAs were purified using 1× Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) and subjected to bisulfite conversion with a ZYMO EZ DNA Methylation-Gold Kit (Zymo research, Irvine, CA, USA). The converted DNAs were then amplified using 25 μl HiFi HotStart U+ RM and 8-bp index primers with a final concentration of 1 μM each. The constructed WGBS libraries were then analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, SantaClara, CA, USA), quantified with a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA,USA), and finally sequenced on an Illumina Hiseq X ten sequencer (PE150 mode) (Illumina, San Diego, CA, USA).