GSM8705590: 140P-IgG-T2; Mus musculus; RNA-Seq
Tumors were excised and processed into single cells first by physical mincing, then enzymatic digestion with collagenase + hyalaronidase + DNAse. Single cell suspension was washed 2x, stained with Sytox Blue and FACS sorted for viable cells. The library construction step of scRNA-seq using 10x Genomics begins with encapsulating individual cells into droplets along with barcoded beads using a microfluidic platform. Cells are lysed, and their RNA binds to barcoded oligonucleotides, ensuring each transcript is tagged with a unique cell barcode and UMI. The captured RNA is reverse transcribed into barcoded cDNA, which is then amplified via PCR. The cDNA is fragmented, end-repaired, A-tailed, and ligated with sequencing adapters. Sample indices are added if multiplexing is required. Finally, the libraries are quality-checked for fragment size and concentration before being sequenced on a high-throughput platform of Illumina.