Identification of Transcription Factor Binding Sites using ATAC-seq

Dendritic cells (DC) are professional antigen presenting cells that comprise different subsets: classical DC type 1 and 2 (cDC1 and cDC2, respectively) and plasmacytoid DC (pDC). In this study cDC1 or pDC were obtained in a two-step in vitro culture system according to Felker et al., J. Immunol. 185, 5326-5335, 2010. Briefly, mouse bone marrow cells were first amplified with a specific cytokine cocktail and then induced to differentiate into DC with Flt3 ligand. cDC1 are CD11c+ CD11b+ XCR1+ and pDC are CD11c+ CD11b- B220+ and thus cDC1 and pDC were obtained by FACS sorting and subjected to Omin-ATAC-seq analysis. As an example of practical application of HINT-ATAC, we performed Omin-ATAC-seq experiments of cDC and pDC cell populations and used HINT-ATAC to detect footprints within ATAC-seq peaks of each of these two cells. Next, we estimated changes in binding activity for all factors with a motif in JASPAR. Cell-specific TF activity is measured by the depth of footprints and number of reads flanking regions. Two known cDC factors, BATF3 and HES1, are detected. In summary, we demonstrated that HINT-ATAC is capable of identifying relevant factors for dendritic cell specification. We performed ATAC-seq experiments in mouse classical dendritic cells type1 (cDC1) and plasmacytoid dendritic cells (pDC) in duplicates. Omni-ATAC-seq was performed according to Corces et al., Nature Methods 14, 959-962, 2017 with minor modifications. Prior to transposition dead cells were removed by centrifugation (800 rpm, 4 min, 4°C). The transposition reaction was with 7.5 μl Tagment DNA Enzyme 1 (TDE1) for 60 min at 37°C. Pre-amplification was with NEBNext Ultra II Q5 Master Mix and Nextera PCR Primers (5 cycles). Quantitative PCR amplification was with NEBNext Ultra II Q5 Master Mix, Nextera PCR Primer, and SYBR Gold to determine the number of additional cycles. PCR amplification of additional cycles was as for pre-amplification. PCR fragments were purified with Qiagen MinElute PCR Purification Kit and library concentration and quality were determined by Agilent High Sensitive DNA Kit and Bioanalyzer, respectively.