ATAC seq of Drosophila accessory glands
All fly stocks were raised in bottles on standard cornmeal media under a controlled 12/12 light cycle regime at 26 C. We performed ATAC-seq experiments on 25 individual accessory glands, using 3 technical replicates. To collect tissue, virgin males were transferred to vials 0-2 hours post eclosion, then dissected for ATAC-seq samples at 48 +/- 2 hours following transfer. Tissues were lysed in 200l of ATAC-seq lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) and manually homogenized 25 times with a plastic pestle, followed by a 1-min incubation on ice, and repeated twice. Following homogenization, samples were pelleted at 4C (100g for 10min) to recover nuclei. We removed the supernatant and washed the pellet in 200l of the lysis buffer. The nuclei preparation was filtered through a 40-m cell strainer and washed with another 200l of lysis buffer. The purified nuclei were isolated by centrifugation at 1,000g for 10min at 4C. After removing the supernatant, 12.5l Nextera Tagment DNA Buffer (Illumina), 11.25l ddH2O and 1.25l Tn5 Transposase were added to the purified nuclei to tagment the DNA. Libraries were then processed using the workflow of Buenrostro et al., 2015 with a final upper and lower size-selection using SPRI beads with bead-to-sample ratios of 0.4 and 1.7, respectively. An aliquot of the purified library was analyzed on a Bioanalyzer (Agilent) to ensure the characteristic nucleosome periodicity of ATAC-seq libraries.