CUT&Tag for H3K4me3 Profiling across Tissues

We used CUT&Tag (17 (link)) in each of the tissue isolations to identify which regions of the genome are marked by trimethylation of histone 3 on lysine 4 (H3K4me3), which correlates with promoter activity. Libraries were prepared as previously described in (30 (link)). Briefly, samples consisting of 100,000 neoblasts, epidermal cells, intestinal cells, or cells from four brains were permeabilized in NE1 buffer (20 mM Hepes-KOH, pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, and 20% glycerol) and fixed in 0.1% formaldehyde for 2 min at 20°C. Samples were incubated with primary antibody rabbit anti-H3K4me3 (Millipore, 07-473) at 4°C overnight. After washing, samples were incubated with secondary antibody guinea pig anti-rabbit (Novusbio) for 1 hour at 20°C, followed by binding of pA-Tn5 for 1 hour at 20°C, and tagmentation in the presence of 10 mM MgCl₂ for 1 hour at 37°C. Tagmented DNA was purified, amplified, and submitted for sequencing. Library preparation for each sample type was executed in triplicate.