Methylation Pattern Analysis Pipeline

For methylation pattern analysis, isolated DNA was sheared using a Bioruptor device (Diagenode). Subsequently, methylated DNA was isolated and eluted in high salt elution buffer using the MethylCap kit (Diagenode) according to the manufacturer´s instructions. Methylated DNA samples were prepared for next generation sequencing using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina according to the manufacturer´s instructions. Amplified libraries were analyzed using the Bioanalyzer 2100 device and DNA 1000 Kit (Agilent) and concentrations were determined by means of Qubit 3.0 fluorometer device and the dsDNA BR assay (Thermo Fisher). Samples were sequenced using the Illumina HiSeq 1500 system (50 bp single reads) in three independent rounds. Sequence images were transformed to BCL files with the Illumina BaseCaller software and samples were demultiplexed to FASTQ files with bcl2fastq v2.17.1.14. Same procedures as above were conducted for quality control, read trimming and adaptor removal. Reads were then mapped to GRCh38 human genome using Bowtie2. Differentially methylated regions were identified using QSEA R package^{56 (link)}.