Background

In eukaryotic organisms, the packaging of long DNA molecules into the nucleus is achieved by wrapping the DNA around histone proteins to form nucleosomes, and further compacting this structure into chromatin (Kornberg, 1974). The control of chromatin condensation is a major player in the regulation of gene expression. Since high DNA compaction restricts access for the transcriptional machinery, closed chromatin is usually associated with repressed gene expression. By contrast, open chromatin is associated with active gene expression, and the presence of regulatory elements like enhancers and promoters (Wegel and Shaw, 2005). The potential to reveal areas of high and low transcriptional activity has led to genome-wide mapping of chromatin accessibility becoming an area of broad interest.

Until now, several technologies for assessing the genomic profile of chromatin compaction have been developed. Some examples are micrococcal nuclease sensitivity (MNase-seq) (Zaret, 2005), formaldehyde-assisted isolation of regulatory elements (FAIRE-Seq) (Giresi et al., 2007), and deoxyribonuclease I hypersensitivity (DNase-Seq) (Boyle et al., 2008). In 2013, Greenleaf and colleagues developed a new method to probe chromatin accessibility, called assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). ATAC-seq is a technique that assesses open chromatin using a mutated hyperactive Tn5 transposase, which simultaneously cuts DNA and tags the resulting fragments with sequencing adaptors, preferentially in decondensed chromatin areas (Buenrostro et al., 2013). DNA sequencing libraries are then generated by PCR amplification and analyzed by high-throughput sequencing. The main advantages of ATAC-seq over the methods mentioned above are the low number of cells required as input and the length of the protocol (see Sun et al., 2019 for a full comparison between several chromatin accessibility assays).

Since development of the ATAC-seq method, the original authors have advanced (Buenrostro et al., 2015) and improved (OMNI ATAC-seq, Corces et al., 2017) the protocol, to overcome some of the initial limitations of this technique. For example, to remove mitochondrial DNA from the transposition reaction, the OMNI ATAC-seq protocol includes a washing step with non-ionic detergent after the cell lysis. Another improvement is the addition of PBS in the transposition mix, to reduce the background noise. Other researchers from different laboratories have also helped to improve and optimize ATAC-seq protocols (Fujiwara et al., 2019), or have adapted them for specific organisms and cell lines (Doganli et al., 2017, Shashikant and Ettensohn, 2019).

Being a core sequencing facility, our group handles a large number of ATAC-seq projects on a regular basis. We have adopted the OMNI ATAC-seq protocol for most of the ATAC-seq experiments, with two main modifications: (i) to handle a large number of projects, cell samples from researchers are sent cryopreserved, to be stored and processed at a convenient time. In this protocol, we include a section focused on the process of thawing cryopreserved cells, which is very important to preserve cell viability, and therefore to the success of an ATAC-seq experiment. (ii) After the purification of the final PCR reaction with the different silica column DNA purification kits available in the market, it is very common to find primer dimers and unwanted DNA fragments, due to over/under transposase digestion or an excess of PCR cycles. Thus, instead of using silica column-based methods, we purify the ATAC-seq libraries with magnetic beads. In addition to the removal of impurities, this allows us to size select the DNA fragments as needed for different samples. The use of magnetic beads for this step also enables the integration of automation platforms for processing a large number of libraries (Hess et al., 2020).

In summary, we present here an ATAC-seq protocol adapted from Corces et al. (2017) that can be used by sequencing core facilities and other laboratories that routinely handle large quantities of samples.