title: GSM6514331: Arabidopsis root cells - sc_43; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: For each sample, ~0.5cm root tips were harvested from 1000-3000 roots and placed into a 35mm petri dish containing a 70 μm cell strainer and 4.5mL enzyme solution (1.5% [w/v] cellulase [ONOZUKA R-10, GoldBio], 0.1% Pectolyase [Sigma P3026], 0.4 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 10 mM CaCl2, 0.1% bovine serum albumin, and 0.000194% (v/v) -mercaptoethanol). The digestion was incubated on an 85 rpm shaker at 25°C for one hour with additional stirring every 15-20 minutes. The resulting cell solution was filtered twice through 40 μm cell strainers and centrifuged for 5 minutes at 500g in a swinging bucket rotor. The pellet was washed with 2mL washing solution (0.4 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 10 mM CaCl2, 0.1% bovine serum albumin, and 0.000194% (v/v) -mercaptoethanol), centrifuged again at 500g for 3 minutes, and the pellet resuspended in washing solution at a concentration of ~2000 cells/uL. We loaded 16,000 cells, with the aim to capture 10,000 cells per sample with the 10X Genomics Chromium 3` Gene expression v3 or v3.1 kits. Cell barcoding and library construction were performed following the manufacturer's instructions. cDNA and final library quality were verified using a Bioanalyzer High Sensitivity DNA Chip (Agilent) and sequenced on an Illumina NovaSeq 6000 instrument to produce 100bp paired-end reads.