CUT&RUN sequencing.

A standard CUT&RUN sequencing protocol was used (41) with minor modifications. Briefly, isolated mDCs from ECs and HIVNs were bound to concanavalin A–coated magnetic beads (Bangs Laboratories), followed by cell permeabilization using 0.01% digitonin (Millipore) in wash buffer. Primary antibodies against intracellular H3K27ac, H3K27me3, and H3K4me3 histone marks (Cell Signaling Technology) were added and cells were incubated at 4°C for overnight. Guinea pig anti-rabbit IgG antibody (Antibodies-online) was used as a negative control. The next day, fusion protein CUTANA pA/G-MNase (20×) (Epicypher) was added and cells were incubated at 4°C for 1 hour, followed by chromatin digestion using 100 mM CaCl₂ (Thermo Fisher Scientific). Stop buffer was then added to stop the chromatin digestion. DNA fragments were extracted using phenol chloroform (Invitrogen) and the extracted DNA was eluted in 1 mM Tris-HCl pH 8.0 (Thermo Fisher Scientific) + 0.1 mM EDTA (Sigma-Aldrich). The DNA library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina Sequencing (New England Biolabs). The DNA library concentration and quality were measured by Qubit 1× dsDNA HS kit (Life Technologies) and D1000 High Sensitivity TapeStation (Agilent), respectively. The sequencing was performed using NextSeq 500/550 High Output v2.5 kit (75 cycles) (Illumina) on a NextSeq 500 Instrument (Illumina). Adapters and low-quality reads were trimmed using Trimmomatic (76) and aligned to the human genome (GRCh38) using Bowtie2 (77). Peak calling was implemented using MACS2 (78). For visualization, the coverage profile was calculated using DeepTools (79). Differential binding analysis was performed using DiffBind (80). The CUT&RUN sequencing data are available in Gene Expression Omnibus accession GSE167563.