ATAC-seq regulatory element types such as promoters and enhancers.

ATAC-seq reportMain aimThe ATAC-seq assay is mainly aiming atidentifying the accessible DNA regions and then interpret the interactionbetween genomic locations of open-chromatin, individual nucleosomes,nucleosome-free region and DNA-binding proteins.

 IntroductionATAC-seq is a technology which can be usedto investigate the chromatin accessibility.In human cells, the long DNA strands arealways being packaged by wrapping it around histones and then further condensedinto chromatin. The ATAC-seq can utilizing the transposase Tn5 which act on thegenomic DNA, cleaving the open chromatin region specifically. By using the NGSsequencing we can easily get the information about the chromatin accessibilityand nucleosome positioning. Procedure:1.

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Cell preparation:In this step, ATAC-seq using the same meansof methods for cell separation and isolation, including cell sorting, cellcollecting, cell lysing (e.g. cold lysis buffer).

2. Transposition reaction:The key step of ATAC-seq procedure istransposition. The adaptor-loaded transposase Tn5 can act preferentially on thenucleosome-free region or the exposed DNA, then catalyze both the movement ofadaptors to the regions and the fragmentation process, this progression is alsocalled “tagmentation”.The natural transposases have low reactiveefficiency so they have been previously mutated in order to increase theiractivity significantly. After the transposition, the sequencing adaptors areinserted into accessible regions of chromatin which contribute to the followingPCR amplification and sequencing step.

 3. AnalysisThe ATAC-seq will produce a large amount ofNGS reads that can be mapped on the reference genome. Based on this, we can geta cut count for each genomic position and create a signal with base-pairresolution.The accessible regions during thisexperiment will contain more sequencing reads because they are the region whichthe transposase acts specifically, and then it will form some peaks in thesignal graph.These regions can then be categorized intovarious regulatory element types such as promoters and enhancers.

Further ,theposition of nucleosomes and nucleosome-free regions can be visualized as well.Advantages: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 onlycost 3 hours in total.3.

High-resolution mapping.4. The entire assay and libraryconstruction can be performed in a simple protocol, significantly reduce theerror which take placed in the multistep protocols or many potentiallyloss-prone steps. Main challenge:Analyzing ATAC-seq experiments has a bigchallenge that the datasets which we finally got tend to be large, sparse andbinary. So, some normal single-cell analysis approaches will not appropriate inthis experiment, for instance, the principal component analysis.

 Article: Exhaustion-associated regulatoryregions in CD8+ tumor-infiltrating T cells Main aimThe main aim of this article is to find outregulatory regions associated with T-cells exhaustion in CD8+tumor-infiltrating T cells, and define chromatin accessibility andtranscriptional profiles in tumor-reactive and non-reactive tumor-infiltratingT-cells by combining RNA sequencing (RNA-seq) and Assay for transposaseaccessible chromatin sequencing (ATAC-seq). How is the ATAC-seq being used andconclusionsIn earlier part of the experiments, NFATtranscription factor is investigated to substantially contribute to exhaustionof CD8+ T-cells.1.

genome-wide changes in chromatinaccessibility of in vitro-generated CTLs caused by re-stimulation. To detect what changes happened tochromatin accessibility of in vitro-generated CTLs after re-stimulation. Andcheck the effect of NFAT and other transcription factors in the exhaustionprogram.(Although for histone modifications,ChIP-seq can be performed on small number of cells, ChIP-seq in this experimentwith limited cell numbers has a low technically feasible. Thus, ATAC-seq wasused to probe chromatin accessibility with very few of cells. ATAC-seq can identify accessible regions,and find out enriched known sequence motifs that are bound by specific familiesof transcription factors. The exact type of transcription factors occupyingaccessible regions can be indicated by RNA-seq.)ATAC-seq was performed on in unstimulatedvitro-differentiated CTLs.

Re-stimulation was performed.(1) re-stimulated by ionomycin (Iono) –activated NFAT(2) re-stimulated by PMA + Iono – activatedNFAT, AP-1, NF?B(3) re-stimulated by PMA + anti-CD3 –activated AP-1, NF?B, less activated NFAT.ATAC-seq was performed on re-stimulatedCTLs.A set of about 4,300 regions wereidentified which showed differential accessibility, and classified into fiveclusters. Enrichment level analysis of motifs was performed. Different accessible regions caused bydifferent stimulation condition were identified based on ATAC-seq experiments.

Combined with motif enrichment analysis, showed a series of known transcriptionfactors that may drive gene expression under different stimulation conditions. This part set a foundation of subsequentanalysis of accessible regions in CD8+ tumor-infiltrating T cells. 2. Genome-wide chromatin accessibilityoverview of OT-I (exhausted) and P14 (non-exhausted) TILs.ATAC-seq was performed on OT-I and P14TILs.1,864 regions that more accessible in OT-Ithan 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 ahigher accessibility in OT-I, and found that these regions showed enrichmentfor 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 accessibleregions specific of OT-I TILs is Nur77.The working hypothesis “NFAT bindingwithout AP-1 contributes to exhaustion of T-cells” and the result of experimentreach an agreement. 3. Identification of specific accessibleregions of exhaustion in OT-I TILs.ATAC-seq was promoted on the 1864 regionsthat have a higher accessibility in OT-I than in P14, 1,414 of them showeddifferent 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 relatedto activation.

By contrast, other 450 regions were defined as exhaustionassociated.