GSM8182739: CO_02; Rattus norvegicus; RNA-Seq
Under sterile conditions, fresh rat brain tissue is first placed in pre-cooled phosphate-buffered saline (PBS) and transferred to a sterile workbench for processing. Then, the tissue is digested with PBS containing 0.25% trypsin and 0.1% DNAse I, gently shaking in a 37°C water bath for 15-30 minutes, with the time adjusted based on the actual digestion results. To terminate digestion, an equal volume of DMEM culture medium containing 10% fetal bovine serum (FBS) is added, and the mixture is filtered through a 40-70μm cell strainer to remove undigested tissue and cell clumps. Subsequently, the cells are centrifuged at 1300 rpm at 4°C for 5 minutes and resuspended in pre-cooled PBS, repeating the washing until the supernatant is clear. Cell density is measured using a cell counting plate or an automatic cell counter, and cell viability is assessed using a 0.4% trypan blue exclusion test, ensuring a viability rate of over 90%. This series of meticulous operations aims to obtain high-quality single-cell suspensions from rat brain tissue, laying the foundation for subsequent single-cell transcriptome analysis. The key technology of the 10x Genomics Chromium™ system leverages millions of unique Barcodes to tag different samples (long DNA molecules/single cells). Initially, Gel beads containing Barcode sequences are mixed with a mixture of samples and enzymes, then combined with an oil surfactant to form GEMs (Gel Bead-In-Emulsions, meaning oil droplets encapsulating Gel beads, samples, and enzyme mixture). The GEMs are collected and flow into a reservoir, where Gel beads dissolve to release Barcode sequences, starting the sample tagging process. The products containing Barcode information from each droplet are mixed together to construct a standard sequencing library.