GSM7152986: Protoplasts of basal root tips, replicate 2, scRNAseq; Phyllostachys edulis; RNA-Seq
The basal root tips (~2 cm) were washed clean and cut into 1–2 mm strips, which  were added to a sterile petri dish with 25 ml sterile enzyme solution (3% cellulase R-10, 0.7% macerozyme R-10, 0.2% pectinase Y-23, 0.3% hemicellulose, 0.1% BSA, 0.01 M CaCl2, 20 mM MES and 0.6 M mannitol) in a vacuum pump at 23°C for 20-30 min, and then the mixture was moved in an incubator with shaking at 65 rpm/min for 3 h at 23°C to release basal root protoplasts. Those protoplasts were filtered with a 40-µm cell strainer and then centrifuged at a speed of 100 g/min for 2 min at 23°C, and after the supernatant was sucked out, 0.6 M mannitol was added to resuspend all protoplasts. After separating root protoplasts, they needed to be purified by sucrose density gradient centrifugation. The total viability of protoplasts needed to reach above 85%, which was further confirmed with trypan blue staining. The concentration of basal root protoplasts was adjusted to 1000 cells/µL. After the protoplast suspensions were put into the 10 x Chromium instrument, the library was constructed using the 10 x Genomics Chromium Single Cell 3'kit (V3). The specific experimental operation steps were implemented according to the scRNA-seq experimental manual of LC-Bio Technology (Hangzhou, China). The Illumina NovaSeq 6000 was used to sequence the library, which demanded a minimum sequencing depth of 20,000 reads per cell.