GSM8745244: CD45+ cells from the brain tissue; Mus musculus; RNA-Seq
Six- to eight-week-old wild-type mice were injected via the tail vein with Cryptococcus neoformans (H99Tdtomato, expressing the fluorescent protein Tdtomato) to simulate fungal bloodstream infection. Six hours post-infection, the mice were euthanized, and cardiac perfusion with saline was performed to thoroughly remove free cells from the vasculature. Brain tissue was then harvested and prepared into a single-cell suspension. Flow cytometry was used to sort CD45+ leukocytes for subsequent single-cell sequencing. Inject the cell suspension into the SCOPE-chip™ microfluidic chip. Based on the principle of “Poisson distribution”, single cells randomly settle into pre-fabricated microwells within the chip under the influence of gravity, ensuring that each microwell captures only one cell. Millions of barcoding beads carrying unique cell barcodes and molecular tags (UMIs) are added to the microwells. Through optimized design, it is ensured that each microwell contains only one bead coexisting with a single cell. Cell lysis is performed within the chip, releasing mRNA that binds to oligo(dT) sequences on the beads through its poly(A) tail, achieving mRNA capture. Simultaneously, each cell and its mRNA molecules are labeled with unique cell barcodes and UMIs. The beads containing the captured mRNA are collected, and the mRNA is reverse transcribed into cDNA, followed by amplification of the cDNA. The amplified cDNA is then fragmented, and adapter sequences are added to construct a sequencing library compatible with the Illumina sequencing platform. The constructed sequencing library is loaded onto the Illumina sequencing platform for high-throughput sequencing, ultimately obtaining gene expression data at the single-cell level.