Droplet Hi-C reveals cell-type-specific chromatin conformation

1. Introduction

In the first and second in this series of blogs, we described the evaluation of chromatin conformation and DNA methylation in the same single nuclei (Heffel et al.) by applying the single-nucleus methyl-3C sequencing (snm3C-seq3) technique (Lee et al. and Tian et al.), the integration of multimodal chromatin and RNA imaging to evaluate regulatory programs active in the developing brain, and the application of single-cell 3D multiomics to dissect neuropsychiatric risk loci. In the third in this series of blogs, Epigenome Technologies reports on a related study describing a novel technique – Droplet Hi-C – that supports the single-cell analysis of chromatin conformation and the integration of additional molecular modalities that can be simultaneously assessed in the same single cell.

Techniques such as snm3C-seq3 (and the evolved form of Droplet Hi-C) support multimodal epigenetic analysis in single cells; meanwhile, parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or Paired-Tag from Epigenome Technologies represents a commercially-available means to generate joint epigenetic and gene expression profiles at single-cell resolution and detect histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Applying Paired-Tag technology may enable quantum leaps forward in our understanding of development and improve disease management (and more!).

2. The Importance of Chromatin Organization to Normal Development and Tumorigenesis

Chromatin conformation plays a key role in embryonic development by influencing gene expression programs; furthermore, abnormal chromatin conformation and altered gene expression profiles prompt disease development (Xu et al.). Examining chromatin conformation in primary tissues/tumor biopsies via bulk Hi-C suffers from challenges associated with the heterogeneous nature of samples, the lack of cell-type resolution, and the complex nature of data analysis. Single-cell Hi-C methods – low-throughput microwell-based or high-throughput combinatorial-indexing-based – have solved many of these problems; furthermore, advances such as the previously described snm3C-seq3 have also permitted simultaneous single-cell Hi-C analysis alongside other molecular modalities.

Researchers guided by Bing Ren (then at the University of California, San Diego) recently introduced a scalable, accessible, droplet-based single-cell Hi-C method called Droplet Hi-C as an optimized method for high-throughput, single-cell chromatin conformation profiling in droplets, which combines in situ chromosomal conformation capture with a commercially available microfluidic platform (Chang et al.). Droplet Hi-C represents a means of simultaneously capturing the 3D structure from tens of thousands of individual cells in a single experiment; however, the authors also reported the additional refinement of Droplet Hi-C to allow the simultaneous capture of transcriptomic and Hi-C data in single cells, thereby supporting the exploration of the links between chromatin architecture and gene expression in normal tissues and tumors.

3. How Droplet Hi-C Works

• Droplet Hi-C – based upon an in situ Hi-C procedure (Rao et al.) – captures genome-wide spatial proximity between chromatin fibers in formaldehyde-cross-linked cells/nuclei through restriction digestion and ligation
• DNA fragmentation and capture take place employing a commercially available microfluidic platform (10x Genomics single-cell assay for transposase-accessible chromatin kit) where cell-specific DNA barcodes are added to the DNA fragments before sequencing library construction and next-generation sequencing
• The Droplet Hi-C procedure lasts ~10h from fixed cells/nuclei to sequencing libraries
• The ability to process eight samples in parallel enables the simultaneous profiling of ~40,000 cells

4. Mapping cell-type-specific chromatin conformation in the mouse cortex

• The analysis of cortex tissues from eight-week-old mice (~6,250 high-quality single-cell chromatin profiles) demonstrated the feasibility of applying Droplet Hi-C to primary tissues
• Intrachromosomal interactions displayed enrichment in single-cell contact maps despite sparse contact signals
• Comparison with related techniques – including sn-m3C-seq – suggested that Droplet Hi-C reliably and robustly detected chromatin conformation in complex tissue samples
• The positive association of gene expression levels with chromatin interactions within the gene body (Zhang et al.) allowed the clustering and annotation of Droplet Hi-C data
• The RNA-seq data employed derived from a previous study (Xie et al.) that applied Droplet Paired-tag
• This analysis supported the resolution of 20 cell groups, with cell-type annotations similar between Droplet Hi-C and sn-m3C-seq
• Subsequent investigations of A and B compartments at 100kb resolution, chromatin loops at 10kb resolution, and multi-way chromatin interactions revealed the ability of Droplet Hi-C to robustly analyze chromatin architecture and cell-type-specific gene regulation in complex tissues

Beyond Chromatin Conformation Analysis of Heterogeneous Tissues

This third blog article of the series describes the development of Droplet Hi-C and the initial application of this single-cell chromatin conformation profiling methodology to map cell-type-specific chromatin architecture in the mouse cortex, which addresses critical gaps in chromatin analysis of heterogeneous tissues. Overall, Droplet Hi-C supports high-throughput single-cell Hi-C assays with minimal hands-on time and lower costs and the identification of cell-type-specific chromatin structures and their correlation to epigenetic modifications when applied to the adult mouse cortex.

The following blog in this series concludes the description of the study of Chang et al., describing the application of Droplet Hi-C to the study of genomic variations in cancer cells, including chromatin structural aberrations and extrachromosomal DNA heterogeneity. The following blog also reports on Paired Hi-C, which supports the joint profiling of chromatin architecture and transcriptomes in single cells to enable the study of gene expression in relation to 3D genome structure.

Paired-tag represents a complementary analytic platform, creating joint epigenetic and gene expression profiles at single-cell resolution and detecting histone modifications and RNA transcripts in individual nuclei. This advance was developed in the Bing Ren lab; now, Epigenome Technologies provides optimized Paired-Tag kits and services under an exclusive license from the Ludwig Institute for Cancer Research.