Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Abstract

New technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization. However, the sparsity of these measurements and the challenge of integrating multiple binding maps represent significant challenges. Here we introduce scCUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.

Nanobody-tethered transposition allows for multifactorial chromatin profiling at single-cell resolution

“…3A), as well 666 antibody-derived tag (ADT) counts per cell, similar to sensitivity recently demonstrated for cell surface protein quantified alongside a single chromatin mark (Extended Data Fig. 3A) (Zhang et al 2021). We clustered cells using a weighted combination of each modality (Hao et al 2021) and annotated cell clusters based on their patterns of protein expression (Fig.…”

“…Bulk-cell data for the cell culture and PBMC datasets were mapped to the hg38 analysis set using Chromap (Zhang et al 2021), with the –preset atac option set. Output BED files produced by Chromap were sorted, bgzip-compressed, and indexed using tabix for further analysis.…”

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

“…We developed a novel Tn5 fusion proteins coupled with secondary nanobodies 24 targeting either mouse or rabbit primary antibody and thus profile any combination of two histone modifications at the same time in single cells. Furthermore, we improved the CUT&Tag tagmentation and library preparation protocol so that nano-CT requires less input material and provides higher depth per cell than previous scCUT&Tagiterations [14][15][16]23 . We also demonstrate that nano-CT can be combined with scATAC-seq to profile open chromatin simultaneously, altogether profiling three epigenetic modalities.…”

Single-cell Gene Regulation Network Inference by Large-scale Data Integration

“…H3K27ac modification is an active enhancer marker that has been profiled using scCUT&Tag-pro in PBMC (16). We built a reference dataset with active histone modifications including H3K27ac (Fig.…”

“…Chromatin Immunoprecipitation Sequencing (ChIP-seq) (12,13) is a direct way to uncover TRs binding in the genome and determine their target genes at the bulk cell level. While the ChIPseq method usually needs millions of cells as starting material, recently developed techniques such as scCUT&Run (14), scCUT&Tag (15), and scCUT&Tag-pro (16) could successfully generate TR binding profiles at the single-cell level. However, most of the data were generated for histone modifications (HMs) rather than TRs, as they have a better abundance on the genome.…”