Single-Cell RNA Sequencing Workflow

After discarding the supernatant, the remaining sample was re-suspended with PBS (HyClone) into 2×10⁵ cells/mL cell suspension, which was then added into the microwell chip using Singleron Matrix^® Single Cell Processing System. Using the same system, Barcoding Beads with barcode labeling were added to the microwell chip. After cytolysis, the polyT structure at the barcode end of the bead surface was coupled with the mRNA tail polyA sequence to capture mRNA. After the complete combination, the magnetic beads were harvested for cDNA reverse transcription and PCR amplification. The obtained cDNA through amplification was fragmented and connected with the sequencing adaptor. The scRNA-seq library was constructed by using GEXSCOPE^® Single Cell RNA Library Kits (Singleron) according to the manufacturer’s protocol (14 (link)). The single library was diluted to 4 nM, pooled, and sequenced with Illumina novaseq 6000, and the paired-end read was 150 bp.