GSM5627311: w1118_1wk_3 (ATAC-seq); Drosophila melanogaster; ATAC-seq
Hearts were exposed and removed, and transferred to 1ml glass douncer with Nuclei EZ Prep kit detergent. Hearts were sheared with 20 loose and 15 tight strokes, then transferred to eppendorf for remainder of SigmaAldrich Nuclei EZ prep kit instructions. Samples were permeabilized in cold nuclear permeabilization buffer ((0.2% IGEPAL-CA630 (I8896, Sigma), 1 mM DTT (D9779, Sigma), Protease inhibitor (05056489001, Roche), and 5% BSA (A7906, Sigma) in PBS (10010-23, Thermo Fisher Scientific)) for 5 minutes on a rotator at 4°C followed by centrifugation for 5 min at 500g at 4°C. After decanting supernatant, the pellet was resuspended in cold tagmentation buffer ((33 mM Tris-acetate (pH = 7.8) (BP-152, Thermo Fisher Scientific), 66 mM K-acetate (P5708, Sigma), 11 mM Mg-acetate (M2545, Sigma), 16% DMF (DX1730, EMD Millipore) in molecular biology grade water (46000-CM, Corning)) followed by incubation with Tagmentation enzyme (FC-121-1030; Illumina) at 37°C with shaking at 500 rpm for 30 min. Tagmented DNA was purified using MinElute PCR purification kit (28004, QIAGEN). The resulting libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (M0541, NEB) with primer extension at 72°C for 5 minutes, denaturation at 98°C for 30 s, followed by 8 cycles of denaturation at 98°C for 10s, annealing at 63°C for 30s and extension at 72°C for 60s. After purification of amplified libraries using MinElute PCR purification kit (28004, QIAGEN), double sided size selection was performed using SPRIselect beads (B23317, Beckman Coulter) with 0.55X beads and 1.5X to sample volume.