Targeted Bisulfite Sequencing of Differentially Methylated CpGs

Differentially methylated CpGs with an ∆β ≥ ±2% methylation and located in differentially expressed gene regions with a logFC ≥ 0.1 were selected for validation with targeted bisulfite sequencing on the Illumina MiSeq platform^{23 (link)}. NRES and RES DNA samples were bisulfite converted using the Epitect 96 Bisulfite kit (Qiagen, USA) as per manufacturer’s guidelines. Primers were designed with the Methyl Primer Express software (ThermoFisher Scientific). All samples were ensured to have an optimal molarity of 2 nM prior to being loaded onto the MiSeq platform with the V3 600 cycle kit (Illumina, US). Methods. Specific details for primer design and amplicon library preparation are included in Supplementary Methods. Upon retrieving raw sequencing data, Trimmomatic (v.0.35) was used to trim adaptor sequences²⁴ . Reads with phred scores <20 were removed and aligned with Bowtie 2 (v 2.1.0)^{25 (link)}. Methylated and non-methylated CpG signals were extracted to calculate the level of methylation at our sites of interest. Results were analyzed using one-tailed t-tests. Correlation of microarray and sequencing methylation values was assessed with Pearson correlation coefficients.