4.3.1. Extraction of DNA and BS-Seq

WT1, WT2, WT3, RT1, RT2, and RT3 samples were separately ground into fine powder in liquid nitrogen. Genomic DNA was extracted using a DNeasy Plant Mini kit (QianGen, Shanghai, China) following the manufacturer's instructions and then checked on 0.1% agarose gels and a NanoPhotometer spectrophotometer (Implen, Westlake Village, CA, USA). DNA concentrations were determined with a Qubit DNA Assay kit on a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA).

Next, 5.2 µg of qualified DNA spiked with 26 ng of lambda DNA (negative control) was sheared in a Covaris S220 ultrasonicator into random 200–300-bp fragments. The resulting fragments were then subjected to end repair, adenylation, and methyl-treated adapter ligation. Two bisulfite treatments with an EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA) were applied to these fragments to transform non-methylated cytosines into uracil for subsequent base pairing with thymine by PCR. After PCR amplification using KAPA HiFi HotStart Uracil + ReadyMix (2×), the generated BS-seq library was quantified on a Qubit 2.0 fluorometer (Life Technologies) and by quantitative PCR, and insert size was assayed on an Agilent Bioanalyzer 2100 system. Sequencing of the BS-seq library, which generated 125/150-bp paired-end reads, was performed on an Illumina Hiseq 2500 platform followed by Illumina CASAVA pipeline analysis.
