Library Preparation for Genome-Wide DNA Methylation Profiling.

DNA 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. Key features • Provides a comprehensive workflow for whole-genome bisulfite sequencing (WGBS) library preparation without relying on commercial kits. • Enables flexible multiplexing by allowing users to customize index systems during oligonucleotide synthesis. • Offers high-resolution, unbiased DNA methylation profiling at single-base resolution. • Optimized for cost-effective and scalable applications, including high-throughput sample processing. • Compatible with a wide range of DNA inputs and adaptable to different species and experimental designs.