ATAC-seq reportMain aimThe 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. IntroductionATAC-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. Procedure: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 called “tagmentation”.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. 3. AnalysisThe 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 resolution.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 graph.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 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 only cost 3 hours in total.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. Main challenge: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. Article: Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells Main aimThe 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). How is the ATAC-seq being used and conclusionsIn earlier part of the experiments, NFAT transcription factor is investigated to substantially contribute to exhaustion of CD8+ T-cells.1. genome-wide 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.(Although for 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 cells. ATAC-seq can 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 re-stimulated CTLs.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 stimulation conditions. 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 TILs.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.