Extended representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells

DNA methylation of CpG dinucleotides impacts transcriptional activity and genome stability, and is altered in human disease. Whole genome and reduced-representation DNA methylation profiling methods have clarified biological roles of DNA methylation, but do not efficiently survey the vast numbers of noncoding regulatory elements in mammalian genomes. Overall design: Here we present a novel extended representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design draws a balance between expanding coverage of regulatory elements and enriching for informative CpG dinucleotides. Barcoded DNA fragments are pooled prior to bisulfite conversion, allowing multiplex processing, improving consistency, and reducing cost and labor. Linear amplification of single stranded bisulfite-treated DNA enables library generation from fragmented and low input DNA samples. We further adapt the method for single-cell analysis, and apply it to evaluate methylation differences between cell types and individual cells in a population.