ATAC-Seq of Wild Type pDC Transcriptomes (CpG 0h, 2h)

Purpose: The goals of this study are to compare the pDC chromatin structure (ATAC-seq) at steady state and at 2h after CpG activation. Methods: Cells were harvested and frozen in culture media containing FBS and 5% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37°C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al., 2013), with some modifications based on (Corces et al., 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina). Results: Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3'-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cut off of p-value 1e-7, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. For differential analysis, reads were counted in all merged peak regions (using Subread), and the replicates for each condition were compared using DESeq2. Conclusions: Our study represents a detailed analysis of naive and activated pDC chromatin that analyzes the global murine TF reservoir as defined by Hu et al. in the AnimalTFdb (Hu et al. , 2019, Nucleic Acids Res 47, D33-D38). Our results show that pDC activation substantially altered the chromatin structure making it more or less accessible to specific TF families. Overall design: Chromatin profiles of bone marrow-derived Flt3-L FACS purified pDCs from wild type mice that were left naïve or stimulated with TLR9 agonist CpG for 2h.