title: GSM4212550: Root-tip protoplast cells, WT, scRNA-seq, replicate 1; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: 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. Conditions were optimized to ensure the inner vascular cell types were dissociated well (even if this was to some extent at the expense of the outer cell files) by microscopic analysis of the protoplasting at regular intervals. 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 PBS+0,04%BSA to yield an estimated concentration of 1000 cells/μl. Cellular suspensions were loaded on a GemCode Single Cell 3' Gel Bead and Library Kit (V2 chemistry, 10X Genomics) according to the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4212550