SM-NucSeq, Genetic and Epigenetic Sequencing of Nucleosomes with Single-Molecule Real-Time Sequencing.

Despite advances in deciphering chromatin structure and dynamics in recent years, mapping the sequence position and combinatorial modifications of individual nucleosomes, the building blocks of chromatin, has yet to be achieved. In this work, we develop SM-NucSeq: a technology that combines single-molecule immunoaffinity detection with single-molecule real-time (SMRT) sequencing in zero-mode waveguides (ZMWs), which are sub-wavelength well-like structures that confine the depth of the excitation beam to the nanometer scale. We show that SM-NucSeq can detect histone modifications on intact nucleosomes and identify their underlying DNA sequences. We leverage the ZMW chips to load nucleosome/polymerase complexes and detect co-occurring histone modifications with fluorescent antibodies, validating each step on a chromatically resolved micromirror TIRF microscope. Finally, the application of SM-NucSeq reveals that DNA synthesis rates are reduced in nucleosomes compared to the double-stranded DNA control sample and that DNA synthesis rate profiles are histone-modification-dependent.