Chromiumsingle cell gene expression solution

On two separate sets of Arabidopsis root protoplasts on separate days, single
cell RNA-Seq was performed using the 10× scRNA-Seq platform, the Chromium
Single Cell Gene Expression Solution (10× Genomics).

For the initial scRNA-seq experiment was performed using the Chromium Single Cell Gene Expression Solution (10× Genomics), following the manufacturer’s protocol. The MSC was isolated from ten 6-week-old mice (all males). Cells were stained with the anti-mouse antibodies CD31 PE-Cy7, CD45 PE-Cy7 and TER119 PE-Cy7 (Biolegend), Sca-1 PE (eBioscience) and PDGFR-α APC (eBioscience), and 30,000 Lin − Sca-1 + Pdgfr-α + were isolated using a BD Bioscience FACS Aria III Fusion. Cells were washed and resuspended in 250 μl FACS buffer (PBS, 2% FBS, 1 mM EDTA), targeting the required 1000 cells/μl concentration, accounting for a 10–20% loss. We pipetted 9.7 μl cell suspension (concentration of 913 cells/μl, ~ 8800 cells), targeting the recovery of ~ 5000 cells. Single-cell RNA-seq libraries were obtained following the 10× Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3’ v2 Reagent Kit. Libraries were sequenced on the NextSeq 500 v2 (Illumina) instrument using 150 cycles (18 bp barcode + UMI, and 132-bp transcript 3’ end), obtaining ~ 5 × 10⁸ raw reads^{46 (link),47 (link)}.