Adult_Nvectensis_scATAC-seq v1

This protocol describes the steps for isolating single nuclei from whole adult Nematostella vectensis polyps for scATAC-seq using 10x Chromium platform. N. vectensis is a brackish-water sea anemone (Cnidaria) that is maintained in the laboratory in 1/3 artificial sea water (~350mOsm/L), as previously reported1. This nuclei isolation protocol combines a slightly hypertonic buffer (TST lysis buffer, ~380mOsm/L)2 with mechanical dissociation using a dounce homogenizer. The resulting cell suspension is mildly fixed with 0.1% PFA3 to mitigate damage during sorting and then purified from debris and aggregates by FACS. Finally, sorted nuclei are permeabilized using a modified hypotonic OmniATAC lysis buffer4 containing 1/10 the standard detergent concentration and supplemented with 70µM Pitstop2 to increase nucleus permeability to Tn55 . After washing, nuclei are visually inspected under the microscope, counted, and tagmented in bulk prior to single-nuclei encapsulation using 10x Chromium platform. scATAC-seq libraries are subsequently prepared following manufacturer`s instructions.