Total time: ~3

Total time: ~3.5 h to 1 1 day. with the thymidine analog bromodeoxyuridine (BrdU) to label newly synthesized DNA. After cell permeabilization, the target proteins are sequentially bound by antibodies and protein A-fused transposase (pA-Tn5), which digests and tags genomic DNA of interest once activated by magnesium. The strand specificity is usually preserved through oligo-replacement. Finally, the producing double-strand DNA is usually denatured and immunoprecipitated with antibodies against BrdU 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 to option methods, the eSPAN protocol is simple, cost-effective and sensitive, even in (S)-(-)-5-Fluorowillardiine a relatively small CXCR4 number of mammalian cells. The whole procedures from cell collection to generation of sequencing-ready libraries can be completed in 2 days. EDITORIAL SUMMARY This protocol explains a detailed workflow for performing genome-wide analyses of strand-specific enrichment of chromatin-associated proteins or altered histones on replicating DNA in low numbers of mammalian cells. PROPOSED TWEET A new protocol for an optimized strand-specific profiling of chromatin-associated proteins and altered histones on replicating DNA PROPOSED TEASER Strand-specific profiling of replicating chromatin Introduction During mitotic cell division, both genetic and epigenetic information is usually propagated into child cells to maintain (S)-(-)-5-Fluorowillardiine the integrity of both the genome and epigenome. Throughout S phase of the cell cycle, DNA replication initiates from replication origins and proceeds bi-directionally with DNA polymerase (Pol (S)-(-)-5-Fluorowillardiine ) synthesizing leading strands constantly and polymerase (Pol ) replicating lagging strands discontinuously1. This inherent asymmetry at the DNA replication fork dictates that different protein machineries are involved in leading and lagging strand DNA synthesis. In eukaryotic cells, DNA synthesis is also tightly coupled to the assembly of replicating DNA into nucleosomes, the first and fundamental step for inheritance of chromatin structure, the establishment of sister chromatid cohesion, and DNA mismatch repair2C4. Therefore, understanding chromatin replication in a strand-specific manner will have a profound impact on chromatin replication and its coupled cellular processes. Several years ago, we developed the eSPAN (enrichment and sequencing of protein associated nascent DNA) method in budding yeast that allowed for the first time to measure the (S)-(-)-5-Fluorowillardiine relative amount of proteins of interest at leading and lagging strands of DNA replication forks5. Using this method, we have shown that DNA Pol and Pol are enriched at leading and lagging strands of replication forks, respectively5, further supporting the idea that these two replicative polymerases are involved in synthesizing different child strands1. Moreover, we showed that, under replication stress, PCNA is usually unloaded from lagging strands and the DNA replication check point kinase Rad53 couples leading and lagging strands synthesis to prevent the generation of excessive single-strand DNA (ssDNA)6. Using the same methods, others have shown that different loaders of PCNA function in DNA replication and sister chromatid cohesion during S phase7. Therefore, the application of eSPAN has yielded useful insights into DNA replication and its coupled processes under normal and replication stress conditions. Recently, we as well as others have also used the eSPAN and comparable methods to analyze how parental histones are put together into nucleosomes following DNA replication8C10, the first step for the inheritance of functional chromatin landscapes in eukaryotic cells. As nucleosomes are barriers for the DNA replication machinery, a couple of nucleosomes ahead of the DNA replication fork are quickly disrupted to allow for efficient DNA synthesis4, 11. Following the passage of replication forks, nucleosomes are reassembled through two unique pathways, deposition of newly synthesized histones and the transfer of parental histones to.