GSM5492069: bri TRIPLE; Arabidopsis thaliana; RNA-Seq
Root tips of 6-d-old seedlings were cut and incubated in protoplasting Solution B [1.5% (wt/vol) cellulysin and 0.1% (wt/vol) pectolyase in Solution A (600 mM mannitol, 2 mMMgCl2, 0.1% [wt/vol] BSA, 2 mMCaCl2, 2 mMMes, 10mMKCl, pH 5.5)] for approximately 1 h at room temperature. Cells were filtered through a 70 μm cell strainer and spun down at 200 × g for 6 min; resuspended in Solution A, filtered through a 40 μm cell strainer and stained for live/dead using 4′,6- diamidino-2-phenylindole (DAPI) at 14 μM final concentration. Cells were sorted on a BD Aria II, and protoplasts not containing the DAPI signal were selected for further analysis. Sorted cells were centrifuged at 4°C at 400g and resuspended in solution A to yield an estimated concentration of 1000 cells/μl. Cellular suspensions were loaded on a Chromium Single Cell 3' GEM, Library & Gel Bead Kit (V3 chemistry, 10X Genomics) according to the manufacturer's instructions.