The ATAC-seq assay is mainly aiming at
identifying the accessible DNA regions and then interpret the interaction
between genomic locations of open-chromatin, individual nucleosomes,
nucleosome-free region and DNA-binding proteins.
ATAC-seq is a technology which can be
used to investigate the chromatin accessibility.
In human cells, the long DNA strands are
always being packaged by wrapping it around histones and then further condensed
into chromatin. The ATAC-seq can utilizing the transposase Tn5 which act on the
genomic DNA, cleaving the open chromatin region specifically. By using the NGS
sequencing we can easily get the information about the chromatin accessibility
and nucleosome positioning.
1. Cell preparation:
In this step, ATAC-seq using the same
means of methods for cell separation and isolation, including cell sorting,
cell collecting, cell lysing (e.g. cold lysis buffer).
2. Transposition reaction:
The key step of ATAC-seq procedure is transposition.
The adaptor-loaded transposase Tn5 can act preferentially on the
nucleosome-free region or the exposed DNA, then catalyze both the movement of
adaptors to the regions and the fragmentation process, this progression is also
The natural transposases have low
reactive efficiency so they have been previously mutated in order to increase
their activity significantly. After the transposition, the sequencing adaptors
are inserted into accessible regions of chromatin which contribute to the
following PCR amplification and sequencing step.
The ATAC-seq will produce a large amount
of NGS reads that can be mapped on the reference genome. Based on this, we can
get a cut count for each genomic position and create a signal with base-pair
The accessible regions during this
experiment will contain more sequencing reads because they are the region which
the transposase acts specifically, and then it will form some peaks in the signal
These regions can then be categorized
into various regulatory element types such as promoters and enhancers. Further
,the position of nucleosomes and nucleosome-free regions can be visualized as
1. Low requirements of the input materials: 500-50000
cells are sufficient for this experiment to get a reliable result.
2. High speed: The whole experiment only cost 3 hours in
3. High-resolution mapping.
4. The entire assay and library construction can be
performed in a simple protocol, significantly reduce the error which take
placed in the multistep protocols or many potentially loss-prone steps.
Analyzing ATAC-seq experiments has a big
challenge that the datasets which we finally got tend to be large, sparse and
binary. So, some normal single-cell analysis approaches will not appropriate in
this experiment, for instance, the principal component analysis.
Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating
main aim of this article is to find out regulatory regions associated with
T-cells exhaustion in CD8+ tumor-infiltrating T cells, and define chromatin
accessibility and transcriptional profiles in tumor-reactive and non-reactive
tumor-infiltrating T-cells by combining RNA sequencing (RNA-seq) and Assay for
transposase accessible chromatin sequencing (ATAC-seq).
is the ATAC-seq being used and conclusions
In earlier part of
the experiments, NFAT transcription factor is investigated to substantially contribute
to exhaustion of CD8+ T-cells.
changes in chromatin accessibility of in vitro-generated CTLs caused by re-stimulation.
To detect what
changes happened to chromatin accessibility of in vitro-generated CTLs after
re-stimulation. And check the effect of NFAT and other transcription factors in
the exhaustion program.
histone modifications, ChIP-seq can be performed on small number of cells,
ChIP-seq in this experiment with limited cell numbers has a low technically feasible.
Thus, ATAC-seq was used to probe chromatin accessibility with very few of
identify accessible regions, and find out enriched known sequence motifs that
are bound by specific families of transcription factors. The exact type of transcription
factors occupying accessible regions can be indicated by RNA-seq.)
ATAC-seq was performed on in
unstimulated vitro-differentiated CTLs.
Re-stimulation was performed.
(1) re-stimulated by ionomycin
(Iono) – activated NFAT
(2) re-stimulated by PMA +
Iono – activated NFAT, AP-1, NF?B
(3) re-stimulated by PMA +
anti-CD3 – activated AP-1, NF?B, less activated NFAT.
ATAC-seq was performed on
A set of about 4,300 regions
were identified which showed differential accessibility, and classified into
five clusters. Enrichment level analysis of motifs was performed.
Different accessible regions
caused by different stimulation condition were identified based on ATAC-seq
experiments. Combined with motif enrichment analysis, showed a series of known
transcription factors that may drive gene expression under different
This part set a foundation of
subsequent analysis of accessible regions in CD8+ tumor-infiltrating T cells.
2. Genome-wide chromatin accessibility overview of OT-I (exhausted) and
P14 (non-exhausted) TILs.
ATAC-seq was performed on OT-I and P14 TILs.
1,864 regions that more
accessible in OT-I than in P14 (red dots), and 453 regions more accessible in
P14 than in OT-I (blue dots) were identified.
They mainly focused on the
regions with a higher accessibility in OT-I, and found that these regions
showed enrichment for transcription factor motifs associated with T-cell
activation, i.e. AP-1, NF?B, and NFAT:AP-1.
Motifs for NFAT (without adjacent AP-1) were enriched in specific regions
of OT-I TILs.
The strongest enrichment of
accessible regions specific of OT-I TILs is Nur77.
The working hypothesis “NFAT binding
without AP-1 contributes to exhaustion of T-cells” and the result of experiment
reach an agreement.
3. Identification of specific accessible regions of exhaustion in OT-I
ATAC-seq was promoted on the 1864 regions that have a higher accessibility
in OT-I than in P14, 1,414 of them showed different accessibility in CD8+
T-cells re-stimulated by PMA + Iono or PMA + anti-CD3 in vitro and OT-I TILs,
these 1,414 regions were defined to be related to activation. By contrast,
other 450 regions were defined as exhaustion associated.