2.9 ∣ DNA Methylation
Total DNA was extracted by the methods mentioned previously.
Cytosine-methylated barcodes were ligated to sonicated DNA as per
manufacturer’s instructions. Then DNA fragments were treated twice with
bisulfite using EZ DNA Methylation-GoldTM Kit (Zymo Research). Library
concentration was quantified using a Qubit® 2.0 Flurometer (Life
Technologies, CA, USA) and quantitative PCR. Insert size was analyzed on
an Agilent Bioanalyzer 2100 system. Transcript libraries were sequenced
on an Illumina Hiseq 2500/4000 platform. Image analysis and base calling
were performed with Illumina CASAVA pipeline. Bismark software (version
0.16.3) was used to perform alignments of bisulfite-treated reads to a
reference genome (Krueger & Andrews,
2011). The reference genome was firstly transformed into
bisulfite-converted version and then indexed using bowtie2
(Langmead & Salzberg, 2012). The results
of methylation extractor were transformed into bigWig format for
visualization using IGV browser. The sodium bisulfite non-coversion rate
was calculated as the percentage of cytosine sequenced at cytosine
reference positions in the lambda genome.
Differentially
methylated regions (DMRs) were identified using the DSS software
(Hao et al., 2014;
Park & Wu, 2016;
Wu et al., 2015). Gene Ontology (GO)
enrichment analysis of genes related to DMRs was implemented using the
GOseq R package (Young et al., 2010), in
which gene length bias was corrected. GO terms with corrected P-value
less than 0.05 were considered significantly enriched by DMR-related
genes. KOBAS software (Mao et al., 2005)
was used to test the statistical enrichment of DMR related genes in KEGG
pathways.