Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell

Abstract

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

“…We sequenced 178-181 million paired end reads and obtained a total of 50-60 million high-quality Hi-C contacts (Extended Data Figure 1). As previously observed, these contact maps show that the genome is segregated into: 1) A and B compartments (broadly regions containing more or fewer genes respectively); 2) megabase-scale topologically associating domains (TADs), which have a higher frequency of intra-domain chromatin interactions; and 3) loops where specific genomic regions contact each other, such as loops mediated via CTCF/Cohesin binding [5][6][7][8][9][10][11][12][13][14][15][16] .…”

“…for point X(k∆t), we fitted the function Sk(m) defined in Eq. (13) by (14) with an additional parameter βi(k) > 0. For the fit, we constrain the variable t in the ensemble [0,∆t,...,(2l+1)∆t].…”

“…This led to a total of ni × 4 parameters, that we organized in a matrix Ri associated with trajectory i: (20) where T is the transpose operator, and ri(k∆t) are four dimensional vectors of the parameters (Eqs. 14,15,16,19).…”

“…However, little is known about the multi-scale chromatin dynamics of cis-regulatory elements or how they relate to genome organisation. To probe these dynamics at the single-cell level, one of two complementary methods are typically used: either live-cell imaging 1-4 or single nucleus versions of chromosome conformation capture experiments (such as Hi-C), which reveal the proximity of DNA sequences in different individual fixed cells [5][6][7][8][9][10][11][12][13][14][15][16] . For example, live-cell tracking of enhancers and promoters has revealed fast diffusion (or 'stirring') during transcription 4 , but the mechanisms underlying these changes in enhancer dynamics or how they relate to Hi-C measurements of enhancer-promoter proximity remain unclear.The nucleosome remodelling and deacetylase (NuRD) complex is a highly conserved 1 MDa multisubunit protein complex that we have previously shown clusters in 3D space in the nucleus with active enhancers and promoters 16 .…”

Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

“…This allows us to access information about the functional 3D genome architecture in live cells. Recent methods now allow single-cell 3C to be combined with microscopy [39], although currently only using lateral imaging (i.e. 2D, in 2D spatial dimensions).…”