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.