JBP1-seq
J-Binding Protein 1 Sequencing
JBP1-seq is a method for genome-wide profiling of 5hmC. It relies on the strong affinity of the J-binding protein 1 (JBP1) for glucosylated 5hmC. The method uses a recombinant JBP1 protein with an additional His tag and Avi tag that is biotinylated and subsequently conjugated to streptavidin-coated magnetic beads (Cui et al., 2014). The JBP1-seq workflow involves 4 main steps. 1) The gDNA is tagmented to fragment it and add adapter sequences. 2) The 5hmC sites are glucosylated by T4-beta-GT. 3) The JBP1-magnetic beads are used to enrich for DNA fragments containing beta-glu-5hmC. 4) The enriched library is amplified to add P5 and P7 adapters and barcodes for sequencing.
Advantages:
- Streamlined protocol
Disadvantages:
- Regions that contain high levels of 5hmC can be overrepresented
- Optimal resolution is approximately 50 bp
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661
References:
Cui L., Chung T. H., Tan D., Sun X. and Jia X. Y. JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC. Genomics. 2014;104:368-375