Rabbit anti h3k4me3

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.

CUT&RUN analysis was performed with approximately 2 × 10⁵ naive ESCs for each library. Bead-bound cells were incubated overnight at 4 ^oC with Rabbit anti-H3K4me3 (Millipore, #07-473) or Rabbit anti-H3K27me3 (Millipore, #07-449) antibody diluted in the antibody buffer (EDTA/Digitonin/wash buffer, EMD Millipore, #300410) (1 µg/500 µl). The beads were washed three times and incubated with 200 µl pA-MNase (0.7 ng/µl) for 1 h at 4 ^oC. 2 mM of CaCl₂ was added to activate MNase for 30 min on ice. The reaction was quenched by 2× stop buffer containing 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 µg/ml RNase A (QIAGEN, #19101), 50 µg/ml glycogen (Roche, #10901393001) and 2 pg/ml Drosophila spike-in DNA at 37 ^oC for 10 min. The soluble chromatins were converted to libraries using the NEBNext Ultra^TM II DNA library Prep kit (NEB, #E7645L). Libraries were subject to pair-ed sequencing on the Illumina NovaSeq 6000 instrument at the University of Michigan core facility.

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.

Immunostaining was performed as described previously (Shlevkov and Morata, 2012 (link)). Images were captured with Leica TCS SPE and Zeiss LSM510 Vertical confocal microscopes. Images were processed with Fiji-ImageJ and Adobe Photoshop CS4 software using Student’s t for significance tests. Statistical analyses were performed with Microsoft Excel software. The following primary antibodies were used: rabbit anti-Caspase 3 (Roche, Basilea, Switzerland) 1:50; mouse anti-ß-Galactosidase (Promega, Madison, WI); rat anti-Cubitus interruptus (2A1; Hybridoma Bank, Iowa City, IA) 1:25; mouse anti-Engrailed (4D9; Hybridoma Bank) 1:50; mouse anti-Patched (Apa-1; Hybridoma Bank) 1:50; rabbit anti-H3K4me3 (07-473; Millipore, Billerica, MA) 1:50. Fluorescently labeled secondary antibodies (Molecular Probes, Eugene, OR) were used in a 1:200 dilution. TO-PRO3 (Invitrogen, Carlsbad, CA) was used in a 1:600 dilution to label nuclei; Phalloidin-Cy5 (65906; Sigma, St. Louis, MO) was used in a 1:200 dilution to label the F-actin network (thus the cell membranes).