Determining the 3D genome structure of a single mammalian cell with Dip-C

Abstract

3D genome structure is highly heterogeneous among single cells and contributes to cellular functions. Our single-cell chromatin conformation capture (3C/Hi-C) technique, Dip-C, enables high-resolution (20 kb or ∼100 nm) 3D genome structure determination from single human and mouse cells. Dip-C is robust, fast, cheap, and does not require specialized equipment. This protocol describes using human and mouse brain samples to perform Dip-C, which has also been applied to other tissue types including the human blood and mouse eye, nose, and embryo. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).

Keywords: Bioinformatics; Cell isolation; Flow Cytometry/Mass Cytometry; Genetics; Genomics; Molecular Biology; Neuroscience; Sequence analysis; Sequencing; Single Cell.

Graphical abstract

Figure 1

Representative Bioanalyzer traces for quality control of the chromatin conformation capture (3C/Hi-C) step, using a combination of NlaIII and MboI restriction enzymes on mouse cells (A) Digestion Control. (B) Ligation Control. Both were run on a Bioanalyzer High Sensitivity DNA kit.

Figure 2

Representative flow cytometry diagrams with 2 roughly equivalent gating strategies The minor fraction of particles with double, triple, or even higher DAPI signals (“V450-A”) were aggregates from the Chromatin Conformation Capture step. Both were run on a BD FACSAria flow sorter. The 2 gating strategies arose from personal preferences of different flow cytometer operators, and do not affect the results. Note that we primarily study cells in the G0/G1 phase of the cell cycle; the corresponding gate (e.g., “G1” in (B)) should be adjusted when studying other phases of the cell cycle.

Figure 3

Representative Bioanalyzer traces for titration of Tn5 transposome concentration during the whole-genome amplification (WGA) by tagmentation step (A) Coarse titration of Illumina TDE1 on purified HeLa gDNA. Range for further titration is indicated by a dashed green box. Note that gDNA only gives approximate results because transposition is slightly different between gDNA and lysate. (B) Fine titration of Tn5 transposome from a different vendor (TTE Mix V50 from Vazyme TD501) on nuclei lysate (see troubleshooting 2 for details). Range suitable for sequencing is indicated by a dashed green box ("acceptable”), and the optimal concentration shown by a solid green box (“best”). All were run on a Bioanalyzer High Sensitivity DNA kit.

Figure 4

Example arrangement of Nextera i7 and i5 indices on a 96-well plate

Figure 5

Representative Bioanalyzer traces before and after size selection of a sequencing library (A) Before size selection. (B) After size selection with 0.7 X SPRISelect beads. (C) Similar to (B) but with 0.6 X beads. All were run on a Bioanalyzer High Sensitivity DNA kit.

Figure 6

Representative data from the mouse brain (A) Chromatin contact maps (top) and 3D genome structures (bottom) of 2 representative single cells, an aggregation of 795 single cells, and bulk Hi-C. All samples were adult neurons from the mouse brain (Tan et al., 2021). Unlike bulk Hi-C, single-cell contact maps show a characteristic pattern of random “patchiness”—especially for inter-chromosomal contacts—indicating highly heterogenous chromosome interactions among single cells (e.g., each chromosome territory only borders a few others in each cell). Raw bulk Hi-C data was downloaded from (Jiang et al., 2017) and reanalyzed by (Tan et al., 2021). Contact maps were visualized with Juicebox.js (Robinson et al., 2018). Note that aggregated or bullk data cannot be represented by a single 3D genome structure, because such data contain mutually conflicting contacts (e.g., inter-chromosomal contacts between all pairs of chromosomes) that is physically impossible for a single structure. (B) t-SNE plot of scA/B showing clusters of 3D genome structure types, from the mouse brain.