GSM8654478: TM-1 1 DPA epidermis and fiber nuclei; Gossypium hirsutum; ATAC-seq
Ovules were digested in a enzyme mix of cellulase and macerase in 450 mM mannitol for 15 minutes to release protoplasts from the epidermal layers. The ovules were then strained out and the protoplasts were allowed to continue digesting for an additional hour to remove any remaining cell wall debris. To recover the protoplasts, we used density gradient flotation: samples were centrifuged for 2 minutes at 200 g at 4°C to gently pellet the protoplasts, after which all but ~100 l of the protoplasting buffer was removed, which was mixed with 0.25 volumes chilled Iodixanol (OptiPrep, Sigma-Aldritch, St. Louis, MO) and used to gently resuspend the pellet. 100 l of chilled 450 mM mannitol was layered atop the denser layer containing iodixanol, and the samples were centrifuged at 200g and 4°C, followed by collecting protoplasts from the top layer. Protoplasts were then treated with nuclei isolation buffer and centrifuged, and nucli were washed with 450 mM mannitol,  pelleted, and resuspended in 10 ATAC buffer. Libraries were constructed acording to the manufacturer's instructions using the Chromium NextGEM SingleCell ATACKit v2 (10x)