GSM5946144: root tip, col_halfMS_r1; Arabidopsis thaliana; RNA-Seq
For each genotype, root tips were placed in 15ml of protoplasting solution [1.25% w/v cellulase, 0.3% w/v macerozyme, 0.4M D-mannitol, 20mM MES (pH 5.7), 20mM KCl, 10mM CaCl2, 0.5% w/v BSA, and 5mM β-mercaptoethanol in ultrapure water] and incubated at room temperature for 1 hour on a tube rotator. The solution was subsequently filtered through a 70μm and a 40μm cell strainer (Falcon) to separate protoplasts from undissociated tissue. Cells were centrifuged for 5 minutes at 500 x g in a swinging bucket centrifuge. The pellet was resuspended in 15ml of protoplast resuspension buffer [0.4M D-mannitol, 20mM MES (pH 5.7), 20mM KCl, 10mM CaCl2, and 0.5% w/v BSA in ultrapure water] and centrifuged at 500 x g for 5 minutes. Cells were resuspended in the protoplast resuspension buffer, and filtered through a 35μm cell strainer Snap Cap (Falcon). The final concentration was adjusted to 900-1,200 cells/μl. Protoplasts from two Arabdiopsis accessions were mixed in equimolar quantities and barcoded with a Chromium Controller (10X Genomics). 10X Genomics 3' Single Cell Gene Expression v2/v3 scRNA-seq