to avoid the loss of genomic DNA in individual cells during DNA purification
[54]. This method is useful for the epigenetic study of heterogeneous and/or rare
cell populations. The approach has been applied in mouse ESCs used as a model of
study due to their versatility, allowing identification of epigenetic and cellular
biological heterogeneity [55]. The disadvantage of this procedure is that DNA
may undergo degradation after the bisulfite step. This method has been improved
in combination with single-cell genome and transcriptome sequencing in order to
explore novel correlations between epigenetic modifications and gene expression in
embryonic stem cells. The protocol has been modified with particular regard to
amplification steps. Further, this method was used to study methylated DNA heterogeneity in a cell population in order to understand the role of this epi-mark in a
well-defined developmental process of disease compared to normal conditions [56].
2.1.9 Single-Cell Reduced Representation Bisulfite Sequencing
(scRRBS)
In the scRRBS method, genomic DNA is previously digested with one or multiple
restriction enzymes (e.g. MspI) that identify common sites on CpG islands (CCGG
sites) to produce sequence-specific fragmentation [54]. The fragmented genomic
DNA is then treated with bisulfite and sequenced. This approach has the advantage
of providing genome-wide coverage of CpGs in islands at single-base resolution as
well as coverage of dense areas in CpG methylation. The disadvantage of conventional RRBS is biased sequence selection due to the fact that restriction enzymes cut
at specific sites. In addition, the assay measures 10–15% of all CpGs in the genome
and is not able to discriminate between 5mC and 5hmC. Differences from the
standard method include the biotinylated 5
0 end of universal adapters and 3
0 end of
index adapters and the introduction of rescue steps. In this way, it is possible to
increase DNA sequences by PCR.
2.1.10 Single-Nucleus Methylcytosine Sequencing (snmC-Seq)
snmC-seq is a high-throughput method based on a whole-genome bisulfite sequencing approach to identify differentially methylated regions across thousands of cells
in order to elucidate cellular diversity of complex tissues [57]. This high-throughput
single-cell sequencing technique can be useful to gain insights into epigenetic
alterations and regulation. The method has been applied in human and mouse frontal
cortex neurons to identify specific neuronal clusters through the use of specific
neuronal markers followed by correlation analyses [57]. Each cluster revealed
differentially methylated regions characterized by low mCG in defined neuronal
populations. Technical application on single neuron epigenomic profile therefore
identified distinct mC regions containing crucial information for specific marker
regions and regulatory elements both in human and mouse.
Advanced Assays in Epigenetics
543
[54]. This method is useful for the epigenetic study of heterogeneous and/or rare
cell populations. The approach has been applied in mouse ESCs used as a model of
study due to their versatility, allowing identification of epigenetic and cellular
biological heterogeneity [55]. The disadvantage of this procedure is that DNA
may undergo degradation after the bisulfite step. This method has been improved
in combination with single-cell genome and transcriptome sequencing in order to
explore novel correlations between epigenetic modifications and gene expression in
embryonic stem cells. The protocol has been modified with particular regard to
amplification steps. Further, this method was used to study methylated DNA heterogeneity in a cell population in order to understand the role of this epi-mark in a
well-defined developmental process of disease compared to normal conditions [56].
2.1.9 Single-Cell Reduced Representation Bisulfite Sequencing
(scRRBS)
In the scRRBS method, genomic DNA is previously digested with one or multiple
restriction enzymes (e.g. MspI) that identify common sites on CpG islands (CCGG
sites) to produce sequence-specific fragmentation [54]. The fragmented genomic
DNA is then treated with bisulfite and sequenced. This approach has the advantage
of providing genome-wide coverage of CpGs in islands at single-base resolution as
well as coverage of dense areas in CpG methylation. The disadvantage of conventional RRBS is biased sequence selection due to the fact that restriction enzymes cut
at specific sites. In addition, the assay measures 10–15% of all CpGs in the genome
and is not able to discriminate between 5mC and 5hmC. Differences from the
standard method include the biotinylated 5
0 end of universal adapters and 3
0 end of
index adapters and the introduction of rescue steps. In this way, it is possible to
increase DNA sequences by PCR.
2.1.10 Single-Nucleus Methylcytosine Sequencing (snmC-Seq)
snmC-seq is a high-throughput method based on a whole-genome bisulfite sequencing approach to identify differentially methylated regions across thousands of cells
in order to elucidate cellular diversity of complex tissues [57]. This high-throughput
single-cell sequencing technique can be useful to gain insights into epigenetic
alterations and regulation. The method has been applied in human and mouse frontal
cortex neurons to identify specific neuronal clusters through the use of specific
neuronal markers followed by correlation analyses [57]. Each cluster revealed
differentially methylated regions characterized by low mCG in defined neuronal
populations. Technical application on single neuron epigenomic profile therefore
identified distinct mC regions containing crucial information for specific marker
regions and regulatory elements both in human and mouse.
Advanced Assays in Epigenetics
543