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.