title: Papilio alphenor CUT&RUN: day 5 pupa hindwing
design: We performed CUT&RUN following Meers et al. (2019) with minor modifications. Hindwings were dissected in room temperature (RT) PBS, then enzymatically dissociated in 750 uL TrypLE Select Enzyme (Gibco, USA) diluted to 5X in PBS,  broken up by gently pipetting 20X with a p1000 pipet set to 500 uL. Tissues were allowed to dissociate at 32C / 1200 rpm in a thermomixer for 20 minutes, pipetting 5X every 5 minutes. Cells were harvested by centrifugation at 600 x g for 3 min, washed twice with 1 mL RT wash buffer (WB), then resuspended in 1 mL RT WB. Cells were bound to concanavalin A-bound magnetic beads (Bangs Labs) for 10 min rotating at RT. Beads were separated using a magnetic stand, then immediately resuspended in ice cold antibody buffer (WB + 0.025% digitonin). Permeabilized cells (500 000 per reaction) were aliquoted to 0.2 mL tubes before adding 0.5 ug primary antibody and nutating overnight at 4oC. Washing, digestion, and purification followed Meers et al. (2019). Libraries were constructed  using the NEBNext Ultra II DNA Library Prep kit following the protocol outlined in Liu (2021) Library Prep for CUT&RUN with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645) V.2 (https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-kxygxm7pkl8j/v2). The key differences between this protocol and the manufacturers protocol are lower annealing temperatures during end repair and PCR. Libraries were pooled and sequenced PE50 on a NovaSeq 6000 in a single SP flow cell to yield ~10M read pairs per sample