WGBS Library Preparation from LCM Tissue

DNA was extracted from LCM tissue using the Nucleospin Tissue XS Kit (Macherey–Nagel) with the modifications suggested for LCM. Extracted DNA was quantified by using the KAPA hgDNA Quantification and QC Kit (Roche). DNA was sheared with Covaris to a size of ~ 175 bp. WGBS libraries were prepared using the KAPA HyperPrep Kit (Roche). Libraries were bisulfite converted postligation using the EZ DNA Methylation-Direct Kit (Zymo). Post bisulfite conversion, libraries were amplified 12 cycles. Libraries were sequenced on an Illumina HiSeq using 150-bp paired-end reads. DNA sequence reads were quality trimmed, and adaptor sequences were removed using Trim-Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The reads were aligned to the human reference sequence (GRCh38/hg38) using Bismark in paired-end Bowtie 2 modes. Unaligned paired-end reads were then processed in single-end mode. Read duplicates were removed using Bismark. Methylation was called on paired-end and single-end files and then merged. Differentially methylated sites were confirmed via multiplex PCR and next-generation sequencing of bisulfite converted DNA as previously described [14 (link)].