Single-cell multi-omic profiling of chromatin conformation and DNA methylation

The ability to profile epigenomic features in single cells is facilitating the study of the variation in transcription regulation at the single cell level. Single cell methods have also facilitated the generation of cell-type resolved transcriptomic and epigenetic profiles of lineages derived from complex heterogeneous samples. However, integrating different epigenetic features remain challenging, as many current methods profile a single data type at at time. Furthermore, some epigenetic features, such as 3D genome organization, are intrinsically variable between single cells of the same lineage, so it remains unclear how well these methods may resolve cell-types from complex mixtures. Here we describe a method for profiling 3D genome organization and DNA methylation in single cells. This protocol accompanies Lee et al. (Nature Methods 2019) after peer review to aid potential users in applying the method to their own samples.


Introduction
Three-dimensional genome architecture is a critical feature of gene regulation in metazoan organisms [1][2][3] . Despite the increasing utility of these datasets, most existing chromatin conformation maps are generated from cell lines in vitro or from bulk tissues in vivo [4][5][6][7][8] . While cell line data has enabled a greater understanding of the general principles of chromatin organization, it cannot fully represent the diversity of cell types that arise in vivo. Recent efforts using single-cell "omics" technologies aims to resolve this challenge by generating single-cell data from complex tissues that are then partitioned into the relevant distinct cell types in silico using data dimensionality reduction and clustering algorithms [9][10][11][12] . However, it remains unclear whether single-cell Hi-C profiles will be suitable for partitioning into constituent cell types in vivo.
In contrast to single cell Hi-C data, single-cell DNA methylome datasets enable high-resolution celltype classification, allowing the reconstruction of epigenomic maps from cell types in primary human tissues 13,14 . 3C or HiC methods capture chromatin configuration by performing proximity ligation with restriction digested genomic DNA in crosslinked nuclei 15 . DNA methylation (mC) is fully preserved in the chimeric DNA molecules produced by 3C or HiC. Therefore, it is feasible to jointly

Sera-Mag Solid Phase Reversible Immobilization (SPRI) Beads
Mix Sera-Mag SpeedBeads and transfer 1mL to a 1.5ml tube.
Place SpeedBeads on a magnetic stand until clears and carefully remove the supernatant. Wash the beads twice with 1ml of TE. For each wash, remove the tube from the magnet and mix by inversions.
Resuspend the washed beads in 1ml of TE.
Add 9g of PEG 8000 to a new 50ml sterile conical tube.
Add 10ml of 5M NaCl to the 50ml tube.
Mix until all dissolves into solution.
Add 1ml of resuspended SpeedBeads to the 50ml tube and fill the volume with MB water.
Test against AMPure XP beads using 100bp DNA ladder.

CT Conversion Reagent
Add 7.9 mL M-Solubilization Buffer and 3 mL M-Dilution Buffer to a bottle of CT Conversion Reagent.
Shake vigorously at room temperature to fully dissolve before adding 1.6 mL M-Reaction Buffer.

M-Wash Buffer
Add 288mL 200-proof Ethanol to four bottles of M-Wash buffer. Invert to mix. Make fresh each time.
Extra buffer can be stored at room temperature.
Aliquot to eight 96-well plates.

Random Priming Master Mix
To have sufficient reagents for robotic preparation of 3,072 reactions, prepare 3600 reactions of * Add 400 µl of 10% Triton-X 100 to 8 mL of Lysis buffer to make Lysis buffer with 0.4% Triton-X. Close the M-tube with its cap and invert gently a couple of times to wash the tube and pour it over the cell strainer.
13. Pool both the lysis buffer together (one without Triton-X and the other with 0.4% Triton-X) to make a 0.2% Triton-X solution.
14. Wash the cell strainer with 2 mL of Lysis buffer with 0.2% Triton-X. Discard the cell strainer. Each quadrant of 384-well plate is barcoded with a distinct indexed random primer (Fig. 2). Every two 384-well plates receive a complete set of all eight indexed random primers. Incubate for 5 min at room temperature. Centrifuge for 5 min at 5,000g and discard the 384-Well DNA Binding Plate.