for re-sequencing. The technique has recently been used in studying genes involved
in mouse brain development such as Arhgap27 and Nhlrc1, which have a significant
number of hmC associated with single-nucleotide polymorphisms. EnIGMA is also
important in the study of ESC memory, since cytosine modifications control epigenetic status across the epi-(genome) [51].
2.1.6 Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq)
CAB-Seq can detect 5caC with single-base resolution in DNA. In this technique,
5caC is selectively labelled by 1-ethyl-3(3-dimethylaminoproyl)-carbodiimide
hydrochloride (EDC) by using a xylene-based primary amine [52]. This specifically
labels 5caC but no other modified cytosine. This modified 5caC does not undergo
deamination by bisulfite as does unmodified 5caC and is therefore sequenced as a
cytosine instead of a thymine. However, this technique has a low protection rate of
5caC deamination (about 50–60%) and requires the sequencing of a non-treated
DNA control. The use of this technique involves mouse ESCs, which are characterized by a strongly decreased quantity of 5caC.
2.1.7 5-Formylcytosine Chemical Modification-Assisted Bisulfite
Sequencing (fCAB-Seq)
fCAB-seq [53] is a chemical method to identify 5fC using BS-seq. The technique
exploits the reaction of hydroxylamine with 5fC but is limited by the fact that it is
only useful for base-resolution mapping. In fCAB-seq, 5fC is protected by bisulfitemediated deamination by treatment with O-ethylhydroxylamine. Hydroxylamineprotected 5fC is therefore read as a cytosine instead of a thymine, as in BS-seq. By
comparing data from BS-seq and fCAB-seq, the locations of 5fC from unmodified
cytosine and 5CaC can be differentiated. RRBS for 5fC is based on the chemical
selective reduction of 5fC and subsequent bisulfite conversion. After bisulfite treatment, 5fC is deformylated and subsequently deaminated in uracil, so that it is read as
a thymine and identified as an unmodified cytosine in BS-seq. This method has also
been used in a mouse ESC model in which genomic distribution and the DNA
glycosylase TDG strongly depend on 5fC regulation. 5fC profiling is further associated with 5mC/5hmC oxidation at different gene regulatory elements. In addition,
5fC accumulation correlates with p300 binding at poised enhancers, thus regulating
important epigenetic mechanisms [53].
2.1.8 Single-Cell Bisulfite Sequencing (scBS-Seq)
This genome-wide DNA methylation sequencing method can also be reproduced in
single cells by integrating some of the steps in the standard RRBS protocol in order
542
C. Dell’Aversana et al.
in mouse brain development such as Arhgap27 and Nhlrc1, which have a significant
number of hmC associated with single-nucleotide polymorphisms. EnIGMA is also
important in the study of ESC memory, since cytosine modifications control epigenetic status across the epi-(genome) [51].
2.1.6 Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq)
CAB-Seq can detect 5caC with single-base resolution in DNA. In this technique,
5caC is selectively labelled by 1-ethyl-3(3-dimethylaminoproyl)-carbodiimide
hydrochloride (EDC) by using a xylene-based primary amine [52]. This specifically
labels 5caC but no other modified cytosine. This modified 5caC does not undergo
deamination by bisulfite as does unmodified 5caC and is therefore sequenced as a
cytosine instead of a thymine. However, this technique has a low protection rate of
5caC deamination (about 50–60%) and requires the sequencing of a non-treated
DNA control. The use of this technique involves mouse ESCs, which are characterized by a strongly decreased quantity of 5caC.
2.1.7 5-Formylcytosine Chemical Modification-Assisted Bisulfite
Sequencing (fCAB-Seq)
fCAB-seq [53] is a chemical method to identify 5fC using BS-seq. The technique
exploits the reaction of hydroxylamine with 5fC but is limited by the fact that it is
only useful for base-resolution mapping. In fCAB-seq, 5fC is protected by bisulfitemediated deamination by treatment with O-ethylhydroxylamine. Hydroxylamineprotected 5fC is therefore read as a cytosine instead of a thymine, as in BS-seq. By
comparing data from BS-seq and fCAB-seq, the locations of 5fC from unmodified
cytosine and 5CaC can be differentiated. RRBS for 5fC is based on the chemical
selective reduction of 5fC and subsequent bisulfite conversion. After bisulfite treatment, 5fC is deformylated and subsequently deaminated in uracil, so that it is read as
a thymine and identified as an unmodified cytosine in BS-seq. This method has also
been used in a mouse ESC model in which genomic distribution and the DNA
glycosylase TDG strongly depend on 5fC regulation. 5fC profiling is further associated with 5mC/5hmC oxidation at different gene regulatory elements. In addition,
5fC accumulation correlates with p300 binding at poised enhancers, thus regulating
important epigenetic mechanisms [53].
2.1.8 Single-Cell Bisulfite Sequencing (scBS-Seq)
This genome-wide DNA methylation sequencing method can also be reproduced in
single cells by integrating some of the steps in the standard RRBS protocol in order
542
C. Dell’Aversana et al.