Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility and other factors. Current methods for defining chromatin states cannot measure more than one aspect in a single experiment at single-cell resolution. Here we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), an assay capable of measuring the genome-wide presence of up to three histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq uses recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk-cell and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in human immune cells. We also extend NTT-seq to enable simultaneous profiling of cell surface protein expression and multimodal chromatin states to study cells of the immune system.
