approaches are continuously evolving. One of the latest applications involves the use
of targeted chromatin ligation (TLC), by which it is possible to start from a very small
quantity of biological material [38]. This is especially important when starting
material comes from biopsy specimens of human tissues or rare cell subpopulations.
This alternative method preserves the sample without requiring the numerous steps
involved in conventional ChIP procedures. The TLC technique increases the recovery
of enriched material and improves the efficiency of ChIP using a simplified workflow.
This approach excludes the use of beads by recruiting proximal binding of the
antibody to specific adapters, followed by subsequent amplification of the ligated
chromatin involving fewer steps and much shorter experimental times. This new
application greatly reduces the methodological complexity by increasing the sensitivity of results obtained. These conditions have been optimized over time and more
specific protocols have been developed.
2.1.1 ChIP in Microfluidic Droplets
This microfluidic-based method is another advanced tool able to overcome the
limitations of standard ChIP described above. It involved for the first time the
development of ChIP in droplets based on a microfluidic platform that combines
nanolitre droplets, magnetic beads and tweezers. This innovative technique has
many advantages including compartmentalization within nanolitre droplets,
improved mixing, reduced consumption of samples and reagents, lower assay time
(about 7 h), decreased number of cells required, lower cross contamination and very
high sensitivity/specificity. The assay is very useful for the study of epigenetic
processes/modifications. The protocol [39] was specifically designed to investigate
four different histone modifications, with known epigenetic roles. This integrated
ChIP procedure is performed using a Teflon tube platform. The system consists of a
syringe pump which pushes sequential droplets across the tube toward a magnetic
tweezer, whose activation/deactivation gives the extraction and dispersion of magnetic beads and droplets. The syringe pump permits droplet manipulation and
processing by switching from injection to aspiration mode. The first train of droplets
is made of magnetic beads associated with antibodies and chromatin, washing
buffer, RNase and proteinase K. Magnetic beads are collected by magnetic tweezers
and then extracted, washed in buffer droplets, transferred to the RNase droplet for
RNA digestion and extracted after 5 min incubation. Subsequently, magnetic beads
are dispersed in the proteinase K droplet for protein digestion and heated for 5 min at
70
C to release DNA from digested proteins. Magnetic beads are then discarded, and
a new train of droplets is generated with charge switch magnetic beads, washing
buffer and elution buffer droplets. These last beads are then transferred to the droplet
containing DNA and digested proteins to capture the DNA, before it is extracted,
washed and eluted and finally discarded before qPCR. The sensitivity of the droplet
methodology is, however, highly dependent on an adequate quantity of magnetic
beads as well as chromatin and specific antibodies. The choice of a well-defined
range of starting material is therefore crucial to improve assay performance.
538
C. Dell’Aversana et al.
of targeted chromatin ligation (TLC), by which it is possible to start from a very small
quantity of biological material [38]. This is especially important when starting
material comes from biopsy specimens of human tissues or rare cell subpopulations.
This alternative method preserves the sample without requiring the numerous steps
involved in conventional ChIP procedures. The TLC technique increases the recovery
of enriched material and improves the efficiency of ChIP using a simplified workflow.
This approach excludes the use of beads by recruiting proximal binding of the
antibody to specific adapters, followed by subsequent amplification of the ligated
chromatin involving fewer steps and much shorter experimental times. This new
application greatly reduces the methodological complexity by increasing the sensitivity of results obtained. These conditions have been optimized over time and more
specific protocols have been developed.
2.1.1 ChIP in Microfluidic Droplets
This microfluidic-based method is another advanced tool able to overcome the
limitations of standard ChIP described above. It involved for the first time the
development of ChIP in droplets based on a microfluidic platform that combines
nanolitre droplets, magnetic beads and tweezers. This innovative technique has
many advantages including compartmentalization within nanolitre droplets,
improved mixing, reduced consumption of samples and reagents, lower assay time
(about 7 h), decreased number of cells required, lower cross contamination and very
high sensitivity/specificity. The assay is very useful for the study of epigenetic
processes/modifications. The protocol [39] was specifically designed to investigate
four different histone modifications, with known epigenetic roles. This integrated
ChIP procedure is performed using a Teflon tube platform. The system consists of a
syringe pump which pushes sequential droplets across the tube toward a magnetic
tweezer, whose activation/deactivation gives the extraction and dispersion of magnetic beads and droplets. The syringe pump permits droplet manipulation and
processing by switching from injection to aspiration mode. The first train of droplets
is made of magnetic beads associated with antibodies and chromatin, washing
buffer, RNase and proteinase K. Magnetic beads are collected by magnetic tweezers
and then extracted, washed in buffer droplets, transferred to the RNase droplet for
RNA digestion and extracted after 5 min incubation. Subsequently, magnetic beads
are dispersed in the proteinase K droplet for protein digestion and heated for 5 min at
70
C to release DNA from digested proteins. Magnetic beads are then discarded, and
a new train of droplets is generated with charge switch magnetic beads, washing
buffer and elution buffer droplets. These last beads are then transferred to the droplet
containing DNA and digested proteins to capture the DNA, before it is extracted,
washed and eluted and finally discarded before qPCR. The sensitivity of the droplet
methodology is, however, highly dependent on an adequate quantity of magnetic
beads as well as chromatin and specific antibodies. The choice of a well-defined
range of starting material is therefore crucial to improve assay performance.
538
C. Dell’Aversana et al.