GSM5804934: Transcriptional DMSO control; Arabidopsis thaliana; RNA-Seq
The whole aerial tissue of the Arabidopsis seedlings was harvested and incubated in protoplasting solution (1.5% (wt/vol) cellulose YC, 0.15% (wt/vol) pectolyase, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 0.1% (wt/vol) BSA, 20 mM MES, pH 5.7). After 1 h gentle shaking, cells were filtered through a 70 μm cell strainer and spun down at 100 × g for 6 min. Pelleted protoplasts were resuspended with washing solution (0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 0.1% (wt/vol) BSA, 20 mM MES, pH 5.7), and filtered through a 40 μm cell strainer. The protoplasts were then sorted on a BD FACSAria II based on the GFP signal. Sorted cells were centrifuged at 400g at 4ºC for 5 minutes and resuspended in washing solution to yield an estimated concentration of 1,000 cells/µl. Cellular suspensions were loaded on either a GemCode Single Cell 3′ Gel Bead and Library Kit (V2 chemistry, 10X Genomics) for the control experiment, or a Chromium Single Cell 3' GEM, Library & Gel Bead Kit (V3 chemistry, 10X Genomics) for the treatment experiment according to the manufacturer's instructions. scRNA-seq