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Single-cell bisulfite sequencing (scBS-seq) enables profiling of DNA methylation at single-nucleotide resolution and across all genomic features. It can explore methylation differences between cells in mixed cell populations and profile methylation in very rare cell types, such as mammalian oocytes and cells from early embryos. Here, we outline the scBS-seq protocol in a 96-well plate format applicable to studies of moderate throughput.DNA methylation is extensively reprogrammed during mammalian embryogenesis and germ cell development. Protocols for genome-