Papilio alphenor ATAC-seq: day 5 pupa hindwing
We performed ATAC experiments following Lewis and Reed (2019) and Buenrostro et al. (2013) with minor modifications. Tissues were dissected in room temperature PBS then immediately transferred into ice cold sucrose buffer in 2 mL dounce homogenizers. Tissues were dissociated using 30 - 50 strokes with the tight pestle, then transferred to cold 1.5 mL tubes. Cells and nuclei were pelleted by centrifugation for 5 min at 1000 x g at 4C, then resuspended in 200 uL lysis buffer (wash buffer: 10 mM Tris-HCl pH7.5, 10 mM NaCl, 3 mM MgCl2 plus 0.2% NP-40) and lysed on ice for 5 min. Nuclei were harvested by centrifugation for 5 min at 1000 x g for 5 min and 4oC, then resuspended in 750 uL ice cold wash buffer and counted. Aliquots of 500,000 cells were pelleted for 5 min at 1000 x g and 4oC, then resuspended in transposition mix (25 uL TD buffer, 2.5 uL TDE1 enzyme, 22.5 uL water). Transposition was performed 30 min at 37C shaking at 1000 rpm, then cleaned up using the Zymo DNA Clean and Concentrator 5 kit. Libraries were amplified 10 cycles before double-sided cleanup (0.5X - 1.8X) with SPRI Select Beads (Beckman-Coulter, USA). We sequenced libraries PE50 on a single lane of a NovaSeq X 10B flowcell, for an average of 30M read pairs per sample.