GSM7731135: ER13_Ctr_2; Arabidopsis thaliana; RNA-Seq
Plants were grown 7 days Arabidopsis roots were cut 700 µm above the meristem and placed in an enzyme solution optimized for Arabidopsis (Mannitol 8%, 400 mM, MES 20 mM, KCl 20 mM, CaCl2 40 mM, pH 5.8 with Tris, BSA 100 µg/ml, 1.2% cellulase “Onozuka” R10, 0.4% macerozyme R‐10 (both Yakult Pharmaceutical Industry CO.). Protoplasts were then filtered through a 20-µm cell strainer and transferred to microcentrifuge tubes for centrifugation.Protoplasts were centrifuged for 3 mins at 500 x g and the pellets were washed and resuspended in washing solution twice (Mannitol 8%, MES 20 mM, KCl 20 mM, CaCl2 10 mM, pH 5.8 with Tris, and BSA 100 µg/ml) and used immediately for single-cell RNAseq.An aliquot of protoplasts was stained with trypan blue (0.2% final) and checked on a hemocytometer under the microscope to determine cell viability and concentration before loading into the 10x Chromium. For each experiment, 16,000 cells were loaded in a Single Cell B Chip (10x Genomics). Single-cell libraries were then prepared using the Chromium Single Cell 3´ library kit V3, following manufacturer instructions. Libraries were sequenced with Novaseq 6000 chip SP V2.5 (4 libraries per chip).