A total of 4.8 μg of genomic DNA spiked with 24 ng lambda DNA was fragmented by sonication to 200–300 bp with the Covaris S220 Focused-ultra sonicator (Covaris, Woburn, MA, USA) followed by end repair and adenylation. All of the DNA fragments were ligated to a sequencing adapter in which all of the cytosines were methylated. The DNA fragments were then treated with bisulfite using an EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA, USA). After treatment, the un-methylated cytosine changed into uracil (PCR amplification to T), and the methylated cytosine remained unchanged. Finally, PCR amplification was used to obtain the final DNA library.
After the library was constructed, a Qubit2.0 Fluorometer was used for the preliminary quantification. The inserted fragment length of the library was then detected using an Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA), and the Q-PCR method was used to determine the effective concentration of the library. Qualified libraries were subjected to high-throughput sequencing using an Illumina Genome Analyzer II to generate 150-bp paired-end reads for the methylation profile analysis by Novogene (Beijing, China).
After the library was constructed, a Qubit2.0 Fluorometer was used for the preliminary quantification. The inserted fragment length of the library was then detected using an Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA), and the Q-PCR method was used to determine the effective concentration of the library. Qualified libraries were subjected to high-throughput sequencing using an Illumina Genome Analyzer II to generate 150-bp paired-end reads for the methylation profile analysis by Novogene (Beijing, China).