CUT&TAG of H4K16ac in male heads replicate 2
CUT&TAG assays were performed using CUT&Tag-IT Assay Kit (Active Motif cat no. 53160). Permeabilized tissues were mixed with activated Concanavalin A beads after their resuspension in the wash buffer. We then incubated overnight at 4 C with primary antibodies from Active Motif; H4K16ac (cat no. 39930). Following this, samples were incubated with a diluted Guinea Pig Anti-Rabbit secondary antibody for 1 hour in a nutator at room temperature. The beads were then washed with a Dig-Wash buffer, and assembled pA-Tn5 transposomes were then added and incubated at room temperature for 1 hour in a nutator mixer. Following incubation, washing of the beads was performed using a Dig-300 buffer. Tagmentation was completed by adding a tagmentation buffer that was mixed with Protease Inhibitor Cocktail and 5% Digitonin and then incubating them at 37 C for 80 minutes. In order to halt the tagmentation process and digest DNA fragments, 4.2 l of EDTA, 1.25 l of 10% SDS and  1.1 l of Proteinase K were added to the Tagmentation buffer and incubated for 60 minutes at 55 C. DNA was extracted and then amplified in a 50 l PCR reaction using combination of unique i7 Indexed primer and one i5 Indexed primer, 30 ul of tagmented DNA, 1 ul of 10mM dNTPs, 10ul of 5X Q5 Reaction Buffer and 0.5 ul of Q5 DNA polymerase. The desired fragment size libraries of CUT&TAG were then selected using 1.1X of SPRI Beads to DNA (55l of SPRI were added to 50 l of DNA). The libraries were checked for quality using both Agilent Bioanalyzer for fragment size distribution and qPCR for DNA library concentration, and those that passed minimum quality checks (with at least 2.5 nM in concentration) were sequenced on Illumina NextSeq550 PE150 Medium at the Vienna Biocenter sequencing facility.