2.1.11 Single-Cell Genome-Wide Methylome and Transcriptome
Sequencing (scM&T-Seq)
scM&T-seq is a recently developed method for the study of parallel single-cell
genome-wide methylome and transcriptome sequencing, which provides information on transcriptional and epigenetic heterogeneity [58]. scM&T-seq uses Smartseq2 and scBS-Seq to determine DNA methylation patterns. This approach is useful
because there is no need to mask coding sequences during analysis since DNA and
RNA are independently amplified. However, the technique has two main drawbacks:
(1) it is impossible to distinguish between 5mC and 5hmC and (2) Smart-seq2 is not
strand-specific and is applicable to only poly(A)-RNA. This technique provides a
very important approach to the analysis of cellular plasticity and heterogeneity.
Applications involve the use of mouse ESCs characterized by a strong heterogeneity
state and variability. Using scM&T-seq it was possible to study these conditions at
single-cell level by NANOG-low and REX1-low cells in sorted populations.
NANOG and REX1 are in fact transcriptional factors involved in ESC differentiation, pluripotency and renewal [58], and this type of approach is of crucial importance to detect methylated distal regulatory elements associated with transcription of
these key pluripotency genes.
2.1.12 Three-Dimensional (3D) Chromatin Methods
The architecture of chromatin is characterized by various levels of complexity that
define the fate of specific genes in terms of regulation and expression, as well as
regulatory regions. Thus, understanding the association of chromatin with specific
proteins such as histones through 3D interactions is highly desirable. Many cuttingedge techniques have been developed to decipher specific genome areas as a “photo
shot”. These technologies involve chromosome conformation capture (3C) and
3C-based methods which decode the 3D organization of the genome and its structural conformation at specific loci. The 3C and 3C-derivative methods [59] offer the
advantage of studying the genome from specific areas up to its entirety both in terms
of complexity and function. The steps of these methods include those already used in
standard ChIP assays, but unlike ChIP, they are able to identify ligation junctions
between genomic loci. The protocol involves chemical cross-linking with formaldehyde and subsequent chromatin purification. Subsequently, specific restriction
enzymes are used that are able to recognize interactions in DNA. Once chromatin
fragments are released, they are further ligated and cross-linking is reversed. The
final step is real-time PCR analysis, which recognizes the ligase product from the
previously isolated DNA, providing a quantitative response of the interaction frequency between two ligated DNA regions. One of the techniques deriving from 3C
is 4C, which allows the detection of an unknown region of DNA that interacts with a
specific locus of interest. Unlike the 3C method, 4C uses a second restriction
544
C. Dell’Aversana et al.
Sequencing (scM&T-Seq)
scM&T-seq is a recently developed method for the study of parallel single-cell
genome-wide methylome and transcriptome sequencing, which provides information on transcriptional and epigenetic heterogeneity [58]. scM&T-seq uses Smartseq2 and scBS-Seq to determine DNA methylation patterns. This approach is useful
because there is no need to mask coding sequences during analysis since DNA and
RNA are independently amplified. However, the technique has two main drawbacks:
(1) it is impossible to distinguish between 5mC and 5hmC and (2) Smart-seq2 is not
strand-specific and is applicable to only poly(A)-RNA. This technique provides a
very important approach to the analysis of cellular plasticity and heterogeneity.
Applications involve the use of mouse ESCs characterized by a strong heterogeneity
state and variability. Using scM&T-seq it was possible to study these conditions at
single-cell level by NANOG-low and REX1-low cells in sorted populations.
NANOG and REX1 are in fact transcriptional factors involved in ESC differentiation, pluripotency and renewal [58], and this type of approach is of crucial importance to detect methylated distal regulatory elements associated with transcription of
these key pluripotency genes.
2.1.12 Three-Dimensional (3D) Chromatin Methods
The architecture of chromatin is characterized by various levels of complexity that
define the fate of specific genes in terms of regulation and expression, as well as
regulatory regions. Thus, understanding the association of chromatin with specific
proteins such as histones through 3D interactions is highly desirable. Many cuttingedge techniques have been developed to decipher specific genome areas as a “photo
shot”. These technologies involve chromosome conformation capture (3C) and
3C-based methods which decode the 3D organization of the genome and its structural conformation at specific loci. The 3C and 3C-derivative methods [59] offer the
advantage of studying the genome from specific areas up to its entirety both in terms
of complexity and function. The steps of these methods include those already used in
standard ChIP assays, but unlike ChIP, they are able to identify ligation junctions
between genomic loci. The protocol involves chemical cross-linking with formaldehyde and subsequent chromatin purification. Subsequently, specific restriction
enzymes are used that are able to recognize interactions in DNA. Once chromatin
fragments are released, they are further ligated and cross-linking is reversed. The
final step is real-time PCR analysis, which recognizes the ligase product from the
previously isolated DNA, providing a quantitative response of the interaction frequency between two ligated DNA regions. One of the techniques deriving from 3C
is 4C, which allows the detection of an unknown region of DNA that interacts with a
specific locus of interest. Unlike the 3C method, 4C uses a second restriction
544
C. Dell’Aversana et al.