Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Abstract

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq

“…Its application prior to next-generation sequencing (NGS) saves time and money. To develop such a control that could also be applied in Arabidopsis organs in which the chromatin accessibility profile is unknown, we designed a qPCR-based approach to define ATAC-seq library quality [37] by assessing the enrichment of accessible loci versus inaccessible loci (Fig. 2).…”

“…In addition, we present a rapid qPCR assay for assessing the quality of ATAC-seq libraries prior to high-throughput sequencing. This assay conserves resources and is particularly valuable when calibrating ATAC-seq for novel organisms or cell types [37]. Notably, the sites we designed to use as positive or negative controls based on available data were found to be accessible in more recent ATAC-seq experiments in various tissues [16, 22, 25].…”

ATAC-seq with unique molecular identifiers improves quantification and footprinting

“…6a), suggesting that many of the additional footprints identified by UMI-ATAC-seq are biologically meaningful. [26][27][28] . However, both published data and our data show an increase in the presumed PCR duplication rates in highly accessible chromatin regions, suggesting that the conventional way to identify PCR duplicates has adverse effects on quantification and footprint identification in ATAC-seq.…”

SPOPmutation confers sensitivity to AR-targeted therapy in prostate cancer by reshaping the androgen-driven chromatin landscape

“…The single-cell suspension was obtained after digesting organoids in TryPLE. ATAC-seq was performed on 100,00 nuclei as previously described (Grbesa et al, 2017) with the minor modifications. Nuclei were isolated in buffer containing 10 mM Tris-Cl pH7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween (Sigma Millipore, Cat # 11332465001), 0.5% NP-40 (Sigma Millipore, Cat # 11332473001), 0.01% digitonin (Promega, Cat# G9441) and 1x protease inhibitors.…”