Recently, the droplet-based microfluidic system was also used by combining DNA
barcoding and NGS in order to collect and compare orthogonal individual cell gene
expression data [40]. This approach is crucial since epigenetic heterogeneity is often
difficult to detect due to the complexity of chromatin organization and different
patterns of variability. Assaf Rotem and colleagues identified the profiling of histone
methylation marks (H3 lysine 4 trimethylation and demethylation) in a mixture of
mouse populations of embryonic stem cells (ESCs), fibroblasts and haematopoietic
progenitor cells by applying this method to discriminate and identify high-quality
chromatin profiles for each cell present in the blend. Droplet-based ChIP assay thus
provides a highly advantageous, rapid and cell-specific approach to epigenetic
analyses.
2.1.2 G4 ChIP-Seq
G4 ChIP-seq is a novel method which can be used to determine G-quadruplex
(G4) structure formation genome-wide in chromatin [41]. DNA sequences are able
to form four-stranded G4 secondary structures that are involved in epigenetic
regulation. This modified protocol applies standard ChIP-seq for the detection of
secondary DNA structures using a specific antibody. As with any ChIP assay, the
quality of the antibody used is critical. This technique uses the G4 structure-specific
single-chain fragment variable antibody BG4, which is prepared using the expression vector pSANG10-3F-BG4.
Although G4 ChIP-seq is a highly innovative method that recognizes chromatin
regions forming G4 DNA structures in a cell population, it presents technical
limitations due to the detection of G4 structures at a given locus present in a
heterogeneous cell population. In addition, this experimental approach is not able
to detect the temporal organization of G4 structure and on which strand it is formed.
The entire protocol requires less than 1 week to be completed and might be also
combined with a microfluidic method (described in the subsection above) for single
cells in order to avoid some of the issues linked to heterogeneous cell populations.
Despite technical limits, the use of this method led to the identification of restricted
regulatory regions of chromatin, focusing specifically on regions regulating cell fate
and function [41] which are not often detectable by standard procedures. The next
step will be to apply this technology in human tissues and other model organisms.
2.1.3 Standard Epigenome-Wide Association Studies (EWAS)
New high-throughput genome-wide epigenetic technologies such as ChIP-seq [42]
and ChIP-on-chip [43] have recently been developed. ChIP-on-chip arrays are
available from Affimetrix, Agilent and NimbleGen, in both whole-genome tiling
array format for model organisms including human and mouse, as well as focused
arrays for promoter regions and custom array designs. The main advantage of ChIPon-chip is the huge amount of data generated which can be statically analysed
Advanced Assays in Epigenetics
539
barcoding and NGS in order to collect and compare orthogonal individual cell gene
expression data [40]. This approach is crucial since epigenetic heterogeneity is often
difficult to detect due to the complexity of chromatin organization and different
patterns of variability. Assaf Rotem and colleagues identified the profiling of histone
methylation marks (H3 lysine 4 trimethylation and demethylation) in a mixture of
mouse populations of embryonic stem cells (ESCs), fibroblasts and haematopoietic
progenitor cells by applying this method to discriminate and identify high-quality
chromatin profiles for each cell present in the blend. Droplet-based ChIP assay thus
provides a highly advantageous, rapid and cell-specific approach to epigenetic
analyses.
2.1.2 G4 ChIP-Seq
G4 ChIP-seq is a novel method which can be used to determine G-quadruplex
(G4) structure formation genome-wide in chromatin [41]. DNA sequences are able
to form four-stranded G4 secondary structures that are involved in epigenetic
regulation. This modified protocol applies standard ChIP-seq for the detection of
secondary DNA structures using a specific antibody. As with any ChIP assay, the
quality of the antibody used is critical. This technique uses the G4 structure-specific
single-chain fragment variable antibody BG4, which is prepared using the expression vector pSANG10-3F-BG4.
Although G4 ChIP-seq is a highly innovative method that recognizes chromatin
regions forming G4 DNA structures in a cell population, it presents technical
limitations due to the detection of G4 structures at a given locus present in a
heterogeneous cell population. In addition, this experimental approach is not able
to detect the temporal organization of G4 structure and on which strand it is formed.
The entire protocol requires less than 1 week to be completed and might be also
combined with a microfluidic method (described in the subsection above) for single
cells in order to avoid some of the issues linked to heterogeneous cell populations.
Despite technical limits, the use of this method led to the identification of restricted
regulatory regions of chromatin, focusing specifically on regions regulating cell fate
and function [41] which are not often detectable by standard procedures. The next
step will be to apply this technology in human tissues and other model organisms.
2.1.3 Standard Epigenome-Wide Association Studies (EWAS)
New high-throughput genome-wide epigenetic technologies such as ChIP-seq [42]
and ChIP-on-chip [43] have recently been developed. ChIP-on-chip arrays are
available from Affimetrix, Agilent and NimbleGen, in both whole-genome tiling
array format for model organisms including human and mouse, as well as focused
arrays for promoter regions and custom array designs. The main advantage of ChIPon-chip is the huge amount of data generated which can be statically analysed
Advanced Assays in Epigenetics
539