GSM8186969: 44hAPF Eye ATAC Rep1; Drosophila melanogaster; ATAC-seq
Dissect 16 eyes or 3 brains per sample in Wash+ solution (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM Spermidine and Roche complete Protease Inhibitor Tablet: 1 tablet per 50 mL). Pipet 100ul of dissected tissues in Wash+ solution directly into 100µL 2X Collagenase/Dispase solution. Incubate at 23C for 30 minutes, shaking at 500 rpm. Vortex for 10 seconds at 60% max speed on a benchtop vortexer. Incubate another 10 minutes at 23C, shaking at 500 rpm. Vortex again for 10 seconds at 60% speed. From here, we use the Omni-ATAC Protocol (Corces et al., 2017). Spin down at 800 x g, 5 minutes, at 4C. Remove supernatant and wash in 200 µL 1X PBS. Repeat 4C spin and remove supernatant. Resuspend cell pellet in 50 µl cold ATAC-RSB (10mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2) supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. Pipette up and down 3 times. Incubate on ice 3 minutes. Add 1 mL cold ATAC-RSB containing 0.1% Tween-20 without NP40 or Digitonin. Invert tube 3 times. Spin down at 800 x g for 10 minutes, 4°C. Discard supernatant and resuspend nuclei in 50 µL transposition reaction mix: 25 µl 2x TDE1 Buffer, 3.5 µl Tn5 Enzyme, 16.5 µl PBS, 0.5 µL 1% Digitonin, 0.5 µL 10% Tween-20, 5 µL water. Incubate at 37°C for 30 minutes shaking at 1000 rpm.Purify using a Qiagen MinElute kit according to manufacturer's protocol. Elute in 21 µl Elution Buffer (10 mM Tris buffer pH 8.0). Library amplification was performed with NEBNext High-Fidelity 2X PCR Master Mix, according to cycling conditions described in Corces et al., 2017, and qPCR side reactions to optimize the number of PCR cycles for each sample. Libraries were assessed on an Agilent Bioanalyzer prior to sequencing