Hiseq x ten sequencing instrument

DNA (50 to 100 ng) from the LCM samples was treated with bisulfite using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA, USA). WGBS libraries were prepared using the TruSeq DNA Methylation Library Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Library quality was evaluated using an Agilent 2100 Bioanalyzer and High-Sensitivity DNA Kit (Agilent, CA, USA). Paired-end sequencing was performed using HiSeq X Ten sequencing instrument (Illumina), yielding sequencing reads approximately 150 bp in size. For data analysis, raw fastq files were trimmed using Trim Galore (version 0.6.5) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, accessed on 26 August 2019) with a cutoff of q30. Thereafter, the trimmed whole-genome sequence fastq files were aligned with the human reference genome 19 (hg19), and duplicate reads were removed using Bismark (version 0.22.3) [16 (link)]. After data preprocessing and mapping, Qualimap (version 2.2.1) [17 (link)] was used to determine the mapping rate, duplication rate, mean coverage, and median insert size. Bismark was also used to determine CpG methylation. For identification of differentially methylated regions (DMRs), methylKit (version 1.20.0) [18 (link)] in R packages was used, with default parameter settings.

We used the QIAamp DNA Mini Kit (Qiagen, Carlsbad, CA, USA) to isolate gDNA from HMEC cultures. The quantity of the extracted gDNA was analyzed with an ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For WGS library construction, we used the TruSeq DNA library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. For WGS, paired-end sequencing was performed on the Illumina HiSeq X Ten sequencing instrument, yielding ~150-bp short sequencing reads.