Protoplast Isolation and scRNA-seq

A commercially available droplet-based system from 10X Genomics Inc. (Zheng et al., 2017) was used with protoplasts isolated essentially according to manufacturer’s instructions (https://assets.ctfassets.net/an68im79xiti/RjkzNZ8LQsGYM2cyGK4g/6afcd76f0a198835e19df7041381c748/CG000090_SamplePrepDemonstratedProtocol_-_Moss_Protoplasts_RevC.pdf) . Briefly, Arabidopsis (Arabidopsis thaliana) seeds were surface sterilized using a 30% (v/v) bleach, 0.1% (v/v) Triton X-100 solution for 10 min and incubated on Murashige and Skoog (MS) growth media covered with 100/47 µm mesh under 16 light (L)/8 dark (D) conditions. At 5 d after germination, the primary root tips were cut and placed into a 35-mm-diameter dish containing a 70-μm strainer and 4 mL enzyme solution (1.25% [w/v] Cellulase [“ONOZUKA” R-10, Yakult], 0.1% [w/v] Pectolyase [P-3026, Sigma-Aldrich], 0.4 M Mannitol, 20 mM MES [pH 5.7], 20 mM KCl, 10 mM CaCl₂, 0.1% [w/v] bovine serum albumin). The dish was rotated at 85 rpm for 1 h at 25°C, and then the cell solution was centrifuged at 500 g for 10 min and the pellet resuspended in 500 μL washing solution (0.4 M Mannitol, 20 mM MES [pH 5.7], 20 mM KCl, 10 mM CaCl₂, 0.1% [w/v] bovine serum albumin). The protoplast solution was strained through a 70-μm filter, and then twice through a 40-μm filter. The filtered solution was centrifuged at 200 g for 6 min, and the pelleted protoplasts resuspended with 30–50 μL washing solution to achieve the desired cell concentration (∼10⁴ protoplasts/mL). The protoplast suspension was loaded into Chromium microfluidic chips with 30 v2 chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 30 v2 reagent kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina HiSeq 4000 according to the manufacturer’s instructions (Illumina).