Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells.

ABSTRACT: Faithful duplication of both genetic and epigenetic information is essential for all eukaryotic cells. DNA replication initiates from replication origins and proceeds bidirectionally but asymmetrically, with the leading strand being synthesized continuously and the lagging strand discontinuously as Okazaki fragments by distinct DNA polymerases. Unraveling the underlying mechanisms of chromatin replication at both strands is crucial to better understand DNA replication and its coupled processes, including nucleosome assembly, sister chromatid cohesion and DNA mismatch repair. Here we describe the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method to analyze the enrichment of proteins of interest, including histones and their modifications at replicating chromatin in a strand-specific manner in mammalian cells. Briefly, cells are pulsed with the thymidine analog bromodeoxyuridine to label newly synthesized DNA. After cell permeabilization, the target proteins are sequentially bound by antibodies and protein A-fused transposase, which digests and tags genomic DNA of interest once activated by magnesium. The strand specificity is preserved through oligo-replacement. Finally, the resulting double-strand DNA is denatured and immunoprecipitated with antibodies against bromodeoxyuridine to enrich nascent DNA associated with proteins of interest. After PCR amplification and next-generation sequencing, the mapped reads are used to calculate the relative enrichment of the target proteins around replication origins. Compared with alternative methods, the eSPAN protocol is simple, cost-effective and sensitive, even in a relatively small number of mammalian cells. The whole procedures from cell collection to generation of sequencing-ready libraries can be completed in 2 days.

SUBMITTER: Li Z 

PROVIDER: S-EPMC9261918 | biostudies-literature |

REPOSITORIES: biostudies-literature