CUT&Tag of H3K14ac/H3K27ac/H4K8ac in Drosophila
CUT&Tag was performed using NovoNGS CUT&Tag 3.0 High-Sensitivity Kit for Illumina (Novoprotein) following the manufacturer's instruction. Each experiment was repeated twice. Twenty animals (at 96 h AEL and 6 h APF) were collected and lysed in nuclear preparation buffer (10mM Tris-HCl (pH7.4), 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween 20, 0.01% Digitonin). Nuclei were harvested and washed using Wash Buffer. The ConA-coated magnetic beads were added per sample and incubated at RT for 10 min. The unbound supernatant was removed, the bead-bound nuclei were resuspended in 50 L Antibody Buffer containing the primary antibody, and the incubation was performed for 2 h at room temperature. The secondary antibody was diluted in 50 L of Antibody Buffer and samples were incubated at RT for 1 h. After removing the liquid, the pAG-Tn5 adapter complex was added to the samples, which was incubated at room temperature for 1 h. Next, samples were re-suspended using Tagmentation buffer and incubated at 37 C for 1 h. To stop tagmentation, SDS was added to samples, which were incubated at 55 C for 1 h. The DNA was purified using Tagment DNA Extract Beads and suspended in water. After amplification by PCR reaction, the DNA was purified using DNA Clean Beads. Libraries were sequenced on illumine Nova-seq platform.