ABSTRACT: Purpose: The goals of this study are to identify BATF binding sites in purified BM-d Flt3-L cultured pDCs (ChIP-seq) at steady state and at 2h after CpG activation. Methods: Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine, and sent to Active Motif Services (Carlsbad, CA) to be processed for ChIP-Seq. In brief, chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (20 µg, spiked-in with 200 ng of Drosophila chromatin) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against BATF (CST, 8638BF). Antibody against H2Av (0.4 ug) was also present in the reaction to ensure efficient pull-down of the spike-in chromatin (Egan et al., 2016). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (QPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad). The resulting signals were normalized for primer efficiency by carrying out QPCR for each primer pair using Input DNA. Results: For ChIP Sequencing Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina’s NextSeq 500 (75 nt reads, single end). Reads were aligned consecutively to the mouse genome (mm10) and to the Drosophila genome (dm3) using the BWA algorithm (default settings). Duplicate reads were removed and only uniquely mapped reads (mapping quality >= 25) were used for further analysis. The number of mouse alignments used in the analysis was adjusted according to the number of Drosophila alignments that were counted in the samples that were compared. Mouse alignments were extended in silico at their 3’-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peak locations were determined using the MACS algorithm (v2.1.0) with a cutoff of p-value = 1e-7. 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. The results were further visualized using Integrative Genomics Viewer (IGV) (Robinson et al., 2011) and modified with Inkscape. Conclusions: Our study represents a detailed analysis of global BATF binding sites in naive and activated pDCs. Our results show that BATF binds not only to promoter regions of genes but extensively to enhancer elements as well. We identify key role players important for pDC biology that are regulated by BATF binding onto their DNA.
