GSM7316288: Isolated germ nuclei prde-1(mj207)V ATAC rep 2; Caenorhabditis elegans; ATAC-seq
Worms were washed with Nuclei Purification Buffer (NPB) (50 mM HEPES pH 7.5, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.1% Tween 20, 0.2 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.25 mM spermine, and 1x Complete Protease Inhibitor Cocktail), and resuspended in 6mL of NPB. Wormed were dounced with fifteen loose strokes were followed by 23 tight strokes. The sample was vortexed twice on medium-high speed for 30 s, followed by 5 min on ice. Worm debris was filtered with three 30μm strainers (MACS) and six 20 μm strainers (Pluriselect). Dapi-stained nuclei were counted using a hemocytometer. Fifty million nuclei were transferred to a 1.5 ml tube for ATAC-seq. ATAC-seq library prepartion was performed as in (Corces et al. 2017). Nuclei was washed with ATAC-Resuspension Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, and 0.01% Digitonin) then spun at 2000rpm for 10 min. Nuclei were then washed with ATAC-Resuspension Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% Tween 20). Fifty million nuclei were resuspended in 50μl transposition mixture, incubated for 30 minutes at 37°C, and then tagmented DNA purified using a  Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D4014). Typically, libraries were amplified using 6–10 PCR cycles.