GSM8012588: Pupal abdominal epidermis, H3K27ac antibody, CUT&RUN, replicate 2; Drosophila melanogaster; OTHER
For each replicate (n=2) we removed abdominal cuticles from 10 pupae 38-45 hAPF. We generated two replicates for the histone H3K27ac antibody (39134, Active Motif) and one control replicate for the Normal Rabbit IgG antibody (Cat. 2729S, Cell Signaling). Both antibodies were used at a 1:100 dilution. After dissection, we washed pupal abdomens by replacing PBS with 1ml of Wash+ buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine with Roche complete EDTA-free protease inhibitor) and centrifuging twice at 12000 g for 5 minutes. We resuspended pupal abdomens in 15 ul of BioMag Plus Concanavalin-A-conjugated magnetic beads (ConA beads, Polysciences Inc.) and followed a CUT&RUN protocol for Drosophila tissues (https://dx.doi.org/10.17504/protocols.io.umfeu3n). Libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB) following  instructions,  but with the following changes: (i) adaptors were diluted 1:10 in water for adaptor ligation (step 2),  (ii) the size selection of the adaptor-ligated DNA in step 3A was omitted (we proceeded directly to step 3B) and (iii) we performed 14 cycles of PCR with 10 seconds of annealing/extension for enrichment of short DNA fragments. Libraries were sequenced on a NovaSeq 6000 system with 150 bp PE reads.