GSM8655316: N1N1 2 DPA epidermis and fiber protoplasts; Gossypium hirsutum; RNA-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 ul 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 ul 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. Libraries were constructed acording to the manufacturer's instructions using the single cell 3' v2 kit (10x Genomics) the the Genome Sequencing and Analysis Facility at UT Austin.