title: GSM8252031: cDC2-WT1-H3k27Ac; Mus musculus; OTHER
library_construction_protocol: 5*10^5 HDAC1-deficient cDC2 were FACS sorted from 3 pooled animals per replicate and genotype (2 replicates per genotype). Prior to FACS sorting, spleens were prepared as mentioned above and were depleted of dead cells/debris and cDC1 by using debris removal and XCR1 microbeads (Miltenyi). Eluted fractions were then enriched with CD11c microbeads resulting in cDC2 population with >80% purity. cDC2s were then FACS sorted to obtain >99% pure cDC2 population (BST2-CD11c+MHCII+XCR1-CD11b+SIRPA+) which was immediately utilized for sample preparation. For CUT and Run, cells were washed once in Buffer 1 (1x eBioscience Permeabilization buffer, 1x EDTA-free Protease Inhibitor cocktail, 0.5mM Spermidine) for 3 minutes at 600 x g and incubated on ice for 1 hour with a 1:200 dilution of H3K27ac antibody (Cell Signaling: 8173S) or rabbit IgG (Cell Signaling; #2729S) in a V-shaped 96-well plate. Next, cells were washed twice with Buffer 1, resuspended in 700 ng/mL pAG/Mnase and incubated on ice for 1 hour. After three washes in Buffer 2 (0.02% digitonin, 0.5mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail in DPBS), pAG/Mnase was activated with 2mM CaCl2for 1 hour on ice. The reaction was quenched with 100µl 2x Stop buffer (Buffer 2 supplemented with 20mM EDTA and4mM EGTA) and incubated for 10 minutes at 37°C. Cells were then spun at 17,000 x g for 5 min and the supernatant was transferred to a fresh tube. DNA was isolated with NEB Monarch PCR and DNA clean up. Libraries were prepared using 1ng using NEBNext® Ultra™ II DNA Library Prep Kit, cleaned up using 1 volume of SPRI beads and subjected to PE150 sequencing. DNA was isolated with NEB Monarch PCR and DNA clean up. Libraries were prepared using 1ng using NEBNext® Ultra™ II DNA Library Prep Kit, cleaned up using 1 volume of SPRI beads and subjected to PE150 sequencing.