FEI-MAN HSUPellegrini, MatteoMatteoPellegriniChen, PaoyangPaoyangChen2026-03-042026-03-042025https://www.scopus.com/pages/publications/105020789323?origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/736079Article number e5488DNA methylation is a fundamental epigenetic mark with critical roles in epigenetic regulation, development, and genome stability across diverse organisms. Whole genome bisulfite sequencing (WGBS) enables single-base resolution mapping of cytosine methylation patterns and has become a standard method in epigenomics. This protocol provides a detailed, step-by-step workflow for WGBS library construction starting from genomic DNA. It includes steps of RNaseA treatment, DNA shearing, end-repair and A-tailing, adapter ligation, bisulfite conversion, library amplification, and quantification. Notably, the method uses self-prepared reagents and customizable index systems, avoiding the constraints of commercial library preparation kits. This flexibility supports cost-effective, scalable methylome profiling, suitable for diverse experimental designs, including high-throughput multiplexed sequencing.trueDNA methylationEpigeneticsEpigenomicsLibrary preparationNext generation sequencingWhole genome bisulfite sequencingjournal article10.21769/bioprotoc.54882-s2.0-105020789323