GSM7986285: Rice root cells - sc_193; Oryza sativa; RNA-Seq
For each sample, ~1 cm root tips were harvested from 15 - 40 roots and placed into a 35 mm petri dish containing a 70 μm cell strainer and 4.5 mL enzyme solution (4% [w/v] cellulase [ONOZUKA R-10, GoldBio], 1.5% Macerozyme R10 [GoldBio], 0.24% 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 28°C for 2.5 hours with additional pipette mixing every 30 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 (HT, 32,000 -> 20,000) 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.