JBP1-seq: A fast and efficient method for genome-wide profiling of 5hmC

We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and next-generation sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylated in vivo and conjugated to magnetic beads via biotin–streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5 h from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling.